AAV&#39;s and uses thereof

ABSTRACT

The invention in some aspects relates to recombinant adeno-associated viruses having distinct tissue targeting capabilities. In some aspects, the invention relates to gene transfer methods using the recombinant adeno-associate viruses. In some aspects, the invention relates to isolated AAV capsid proteins and isolated nucleic acids encoding the same.

RELATED APPLICATIONS

This Application is a continuation application which claims priorityunder 35 U.S.C. § 120 to U.S. application Ser. No. 15/007,559, entitled“NOVEL AAV'S AND USES THEREOF” filed on Jan. 27, 2016, which is acontinuation of U.S. application Ser. No. 14/246,560, entitled “NOVELAAV'S AND USES THEREOF” filed on Apr. 7, 2014, which is a continuationapplication which claims priority under 35 U.S.C. § 120 to U.S.application Ser. No. 13/322,164, entitled “NOVEL AAV'S AND USES THEREOF”filed on Feb. 15, 2012, which is a national stage filing under 35 U.S.C.§ 371 of international application PCT/US2010/032158, filed Apr. 23,2010, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 61/182,084, entitled “NOVEL AAV'S ANDUSES THEREOF” filed on May 28, 2009, the entire content of eachapplication which is incorporated by reference herein.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersDK047757, HL059407, and NS076991, awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention in some aspects relates to isolated nucleic acids,compositions, and kits useful for identifying adeno-associated virusesin cells. In some aspects, the invention provides novel AAVs and methodsof use thereof as well as related kits.

BACKGROUND OF INVENTION

Adenoassociated Virus (AAV) is a small and helper dependent virus. Itwas discovered in 1960s as a contaminant in adenovirus (a cold causingvirus) preparations. Its growth in cells is dependent on the presence ofadenovirus and, therefore, it was named as adeno-associated virus.Before 2002, a total of 6 serotypes of AAVs were identified, includingthe serotype 2 which was the first AAV developed as vector for genetransfer applications and the one used in the recent break through eyegene therapy trials. In the earlier attempts to develop AAV as genetransfer vehicle, prototype AAV vector based on serotype 2 effectivelyserved as a proof-of-concept showcase and accomplished non-toxic andstable gene transfer in murine and large animal models in differenttarget tissues. For instance an 8 year, stable vision improvement wasobserved in a dog model of LCA after a single injection and a 9 year,stable gene expression in Macaque muscle was achieved. However, theseproof-of-concept studies also revealed a significant shortcoming whichis a poor gene transfer efficiency in major target tissues.

Methods for discovering novel AAVs have been largely focused onisolating DNA sequences for AAV capsids, which relate to the tissuetargeting capacity of the virus. To date, the principal methods employedfor identifying novel AAV take advantage of the latency of AAV proviralDNA genomes and focus on rescuing persisted viral genomic DNA. The majorchallenge in DNA-targeted AAV isolation is that the abundance ofpersisted AAV genomes is often very low in most of tissues particularlyin human tissues, which makes AAV rescue unachievable in many cases.

SUMMARY OF INVENTION

The invention in some aspects relates to novel AAVs for gene therapyapplications. In some aspects the invention relates to AAVs havingdistinct tissue targeting capabilities (e.g., tissue tropisms), whichachieve stable and nontoxic gene transfer at the efficiencies similar tothose of adenovirus vectors. In some aspects, the invention relates toisolated nucleic acids (e.g., primers, transgenes), composition and kitsuseful in the methods for identifying novel AAVs.

The invention in some aspects provides an isolated nucleic acidcomprising a sequence selected from the group consisting of: SEQ ID NO:13-86, which encodes an AAV capsid protein. In some embodiments, afragment of the isolated nucleic acid is provided. In certainembodiments, the fragment of the isolated nucleic acid does not encode apeptide that is identical to a sequence of any one of SEQ ID NOs:177-183.

The invention in some aspects provides an isolated AAV capsid proteincomprising an amino acid sequence selected from the group consisting of:SEQ ID NOs: 87-160 and 171-176. In some embodiments, the isolated AAVcapsid protein comprises a sequence selected from the group consistingof: SEQ ID NOs: 147, 148, and 152, wherein an amino acid of the sequencethat is not identical to a corresponding amino acid of the sequence setforth as SEQ ID NOs: 179 is replaced with a conservative substitution.In some embodiments, the isolated AAV capsid protein comprises asequence selected from the group consisting of: SEQ ID NOs: 87-128 and171-178, wherein an amino acid of the sequence that is not identical toa corresponding amino acid of the sequence set forth as SEQ ID NO: 180is replaced with a conservative substitution. In some embodiments, theisolated AAV capsid protein comprises a sequence set forth as SEQ ID NO:156, wherein an amino acid of the sequence that is not identical to acorresponding amino acid of the sequence set forth as SEQ ID NO: 181 isreplaced with a conservative substitution. In some embodiments, theisolated AAV capsid protein comprises a sequence selected from the groupconsisting of: SEQ ID NOs: 149-151, 153-155, and 157-159, wherein anamino acid of the sequence that is not identical to a correspondingamino acid of the sequence set forth as SEQ ID NO: 182 is replaced witha conservative substitution. In some embodiments, the isolated AAVcapsid protein comprises a sequence set forth as SEQ ID NO: 134, whereinan amino acid of the sequence that is not identical to a correspondingamino acid of the sequence set forth as SEQ ID NO: 183 is replaced witha conservative substitution. In some embodiments, the isolated AAVcapsid protein comprises a sequence selected from the group consistingof: SEQ ID NOs: 129-133 and 135-146, wherein an amino acid of thesequence that is not identical to a corresponding amino acid of thesequence set forth as SEQ ID NO: 184 is replaced with a conservativesubstitution. In some embodiments, the isolated AAV capsid proteincomprises a sequence set forth as SEQ ID NO: 160, wherein an amino acidof the sequence that is not identical to a corresponding amino acid ofthe sequence set forth as SEQ ID NO: 185 is replaced with a conservativesubstitution. In certain embodiments, a peptide fragment of the isolatedAAV capsid protein is provided. In one embodiment, the peptide fragmentis not identical to a sequence of any one of SEQ ID NO: 179-185. In someembodiments, an isolated AAV capsid protein comprising the peptidefragment is provided.

In some embodiments, the isolated AAV capsid protein comprises asequence set forth as SEQ ID NO: 179, wherein an amino acid of thesequence is replaced with a non-identical, corresponding amino acid ofthe sequence set forth as SEQ ID NOs: 147, 148, or 152. In someembodiments, the isolated AAV capsid protein comprises a sequence setforth as SEQ ID NO: 180, wherein an amino acid of the sequence isreplaced with a non-identical, corresponding amino acid of the sequenceset forth as any one of SEQ ID NOs: 87-128 and 171-176. In someembodiments, the isolated AAV capsid protein comprises a sequence setforth as SEQ ID NO: 181, wherein an amino acid of the sequence isreplaced with a non-identical, corresponding amino acid of the sequenceset forth as SEQ ID NO: 156. In some embodiments, the isolated AAVcapsid protein comprises a sequence set forth as SEQ ID NO: 182, whereinan amino acid of the sequence is replaced with a non-identical,corresponding amino acid of the sequence set forth as any one of SEQ IDNOs: 149-151, 153-155, and 157-159. In some embodiments, the isolatedAAV capsid protein comprises a sequence set forth as SEQ ID NO: 183,wherein an amino acid of the sequence is replaced with a non-identical,corresponding amino acid of the sequence set forth as SEQ ID NO: 134. Insome embodiments, the isolated AAV capsid protein comprises a sequenceset forth as SEQ ID NO: 184, wherein an amino acid of the sequence isreplaced with a non-identical, corresponding amino acid of the sequenceset forth as any one of SEQ ID NOs: 129-133 and 135-146. In someembodiments, the isolated AAV capsid protein comprises a sequence setforth as SEQ ID NO: 185, wherein an amino acid of the sequence isreplaced with a non-identical, corresponding amino acid of the sequenceset forth as SEQ ID NO: 160. In some embodiments, isolated nucleic acidsencoding any of the foregoing isolated AAV caspid protein are provided.

In certain aspects of the invention, a composition is provided thatcomprises any of the foregoing isolated AAV capsid proteins. In someembodiments, the composition further comprises a pharmaceuticallyacceptable carrier. In some embodiments a composition of one or more ofthe isolated AAV capsid proteins of the invention and a physiologicallycompatible carrier is provided.

In certain aspects of the invention, a recombinant AAV (rAAV) isprovided that comprises any of the foregoing isolated AAV capsidproteins. In some embodiments, a composition comprising the rAAV isprovided. In certain embodiments, the composition comprising the rAAVfurther comprises a pharmaceutically acceptable carrier. A recombinantAAV is also provided, wherein the recombinant AAV includes one or moreof the isolated AAV capsid proteins of the invention.

In some aspects of the invention, a host cell is provided that containsa nucleic acid that comprises a coding sequence selected from the groupconsisting of: SEQ ID NO: 13-86 that is operably linked to a promoter.In some embodiments, a composition comprising the host cell and asterile cell culture medium is provided. In some embodiments, acomposition comprising the host cell and a cryopreservative is provided.

According to some aspects of the invention, a method for delivering atransgene to a subject is provided. In some embodiments, the methodcomprises administering any of the foregoing rAAVs to a subject, whereinthe rAAV comprises at least one transgene, and wherein the rAAV infectscells of a target tissue of the subject. In some embodiments, subject isselected from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig,and a non-human primate. In one embodiment, the subject is a human. Insome embodiments, the at least one transgene is a protein coding gene.In certain embodiments, the at least one transgene encodes a smallinterfering nucleic acid. In certain embodiments, the small interferingnucleic acid is a miRNA. In certain embodiments, the small interferingnucleic acid is a miRNA sponge or TuD RNA that inhibits the activity ofat least one miRNA in the subject. In certain embodiments, the miRNA isexpressed in a cell of the target tissue In certain embodiments, thetarget tissue is skeletal muscle, heart, liver, pancreas, brain or lung.In some embodiments, the transgene expresses a transcript that comprisesat least one binding site for a miRNA, wherein the miRNA inhibitsactivity of the transgene, in a tissue other than the target tissue, byhybridizing to the binding site. In certain embodiments, the rAAV isadministered to the subject intravenously, transdermally, intraocularly,intrathecally, intracererbally, orally, intramuscularly, subcutaneously,intranasally, or by inhalation.

According to some aspects of the invention, a method for generating asomatic transgenic animal model is provided. In some embodiments, themethod comprises administering any of the foregoing rAAVs to a non-humananimal, wherein the rAAV comprises at least one transgene, and whereinthe rAAV infects cells of a target tissue of the non-human animal. Insome embodiments, the at least one transgene is a protein coding gene.In certain embodiments, the at least one transgene encodes a smallinterfering nucleic acid. In some embodiments, the at least onetransgene encodes a reporter molecule. In certain embodiments, the smallinterfering nucleic acid is a miRNA. In certain embodiments, the smallinterfering nucleic acid is a miRNA sponge or TuD RNA that inhibits theactivity of at least one miRNA in the animal. In certain embodiments,the miRNA is expressed in a cell of the target tissue In certainembodiments, the target tissue is skeletal muscle, heart, liver,pancreas, brain or lung. In some embodiments, the transgene expresses atranscript that comprises at least one binding site for a miRNA, whereinthe miRNA inhibits activity of the transgene, in a tissue other than thetarget tissue, by hybridizing to the binding site. According to someaspects of the invention, methods are provided for generating a somatictransgenic animal model that comprise administering any of the foregoingrAAVs to a non-human animal, wherein the rAAV comprises at least onetransgene, wherein the transgene expresses a transcript that comprisesat least one binding site for a miRNA, wherein the miRNA inhibitsactivity of the transgene, in a tissue other than a target tissue, byhybridizing to the binding site of the transcript. In some embodiments,the transgene comprises a tissue specific promoter or induciblepromoter. In certain embodiments, the tissue specific promoter is aliver-specific thyroxin binding globulin (TBG) promoter, a insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatinekinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosinheavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.In certain embodiments, the rAAV is administered to the animalintravenously, transdermally, intraocularly, intrathecally, orally,intramuscularly, subcutaneously, intranasally, or by inhalation.According to some aspects of the invention, a somatic transgenic animalmodel is provided that is produced by any of the foregoing methods.

In other aspects of the invention, a kit for producing a rAAV isprovided. In some embodiments, the kit comprises a container housing anisolated nucleic acid having a sequence of any one of SEQ ID NO: 13-86.In some embodiments, the kit further comprises instructions forproducing the rAAV. In some embodiments, the kit further comprises atleast one container housing a recombinant AAV vector, wherein therecombinant AAV vector comprises a transgene.

In other aspects of the invention, a kit is provided that comprises acontainer housing a recombinant AAV having any of the foregoing isolatedAAV capsid proteins. In some embodiments, the container of the kit is asyringe.

In other aspects, the invention relates to the use of AAV based vectorsas vehicles for, delivery of genes, therapeutic, prophylactic, andresearch purposes as well as the development of somatic transgenicanimal models. In some aspects, the invention relates to AAV serotypesthat have demonstrated distinct tissue/cell type tropism and can achievestable somatic gene transfer in animal tissues at levels similar tothose of adenoviral vectors (e.g., up to 100% in vivo tissuetransduction depending upon target tissue and vector dose) in theabsence of vector related toxicology. In other aspects, the inventionrelates to AAV serotypes having liver, heart, skeletal muscle, brain andpancreas tissue targeting capabilities. These tissues are associatedwith a broad spectrum of human diseases including a variety ofmetabolic, cardiovascular and diabetic diseases. In some embodiments therAAV includes at least one transgene. The transgene may be one whichcauses a pathological state. In some embodiments, the transgene encodinga protein that treats a pathological state.

In another aspect the novel AAVs of the invention may be used in amethod for delivering a transgene to a subject. The method is performedby administering a rAAV of the invention to a subject, wherein the rAAVcomprises at least one transgene. In some embodiments the rAAV targets apredetermined tissue of the subject.

In another aspect the AAVs of the invention may be used in a method forgenerating a somatic transgenic animal model. The method is performed byadministering a rAAV of the invention to an animal, wherein the rAAVcomprises at least one transgene, wherein the transgene causes apathological state, and wherein the rAAV targets a predetermined tissueof the animal.

In one embodiment the rAAV has a AAV capsid having an amino acidsequence selected from the group consisting of: SEQ ID NOs: 87-160 and171-178.

The transgene may express a number of genes including cancer relatedgenes, pro-apoptotic genes and apoptosis-related genes. In someembodiments the transgene expresses a small interfering nucleic acidcapable of inhibiting expression of a cancer related gene. In otherembodiments the transgene expresses a small interfering nucleic acidcapable of inhibiting expression of a apoptosis-related gene. The smallinterfering nucleic acid in other embodiments is a miRNA or shRNA.According to other embodiments the transgene expresses a toxin,optionally wherein the toxin is DTA. In other embodiments the transgeneexpresses a reporter gene which is optionally a reporter enzyme, such asBeta-Galactosidase or a Fluorescent protein, such as GFP.

The transgene may express a miRNA. In other embodiments the transgeneexpresses a miRNA sponge, wherein miRNA sponge inhibits the activity ofone or more miRNAs in the animal. The miRNA may be an endogenous miRNAor it may be expressed in a cell of a heart, liver, skeletal muscle,brain or pancreas tissue, in some embodiments.

In one embodiment the target tissue of an AAV is gonad, diaphragm,heart, stomach, liver, spleen, pancreas, or kidney. The rAAV maytransduce many different types of tissue, such as muscle fibers,squamous epithelial cells, renal proximal or distal convoluted tubularcells, mucosa gland cells, blood vessel endothelial cells, or smoothmuscle cells.

In some embodiments the rAAV is administered at a dose of 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per subject. In some embodimentsthe rAAV is administered at a dose of 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴genome copies per kg. The rAAV may be administered by any route. Forinstance it may be administered intravenously (e.g., by portal veininjection) in some embodiments.

In some embodiments the transgene includes a tissue specific promotersuch as a liver-specific thyroxin binding globulin (TBG) promoter, ainsulin promoter, a glucagon promoter, a somatostatin promoter, apancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, acreatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, aα-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT)promoter.

The somatic transgenic animal model may be a mammal, such as a mouse, arat, a rabbit, a dog, a cat, a sheep, a pig, a non-human primate.

In some embodiments a putative therapeutic agent may be administered tothe somatic transgenic animal model to determine the effect of theputative therapeutic agent on the pathological state in the animal.

In another aspect the invention is a somatic transgenic animal producedby the methods described herein.

A kit for producing a rAAV that generates a somatic transgenic animalhaving a pathological state in a predetermined tissue is providedaccording to another aspect of the invention. The kit includes at leastone container housing a recombinant AAV vector, at least one containerhousing a rAAV packaging component, and instructions for constructingand packaging the recombinant AAV.

The rAAV packaging component may include a host cell expressing at leastone rep gene and/or at least one cap gene. In some embodiments the hostcell is a 293 cell. In other embodiments the host cell expresses atleast one helper virus gene product that affects the production of rAAVcontaining the recombinant AAV vector. The at least one cap gene mayencode a capsid protein from an AAV serotype that targets thepredetermined tissue.

In other embodiments a rAAV packaging component includes a helper virusoptionally wherein the helper virus is an adenovirus or a herpes virus.

The rAAV vector and components therein may include any of the elementsdescribed herein. For instance, in some embodiments the rAAV vectorcomprises a transgene, such as any of the transgenes described herein.In some embodiments the transgene expresses a miRNA inhibitor (e.g., amiRNA sponge or TuD RNA), wherein miRNA inhibitor inhibits the activityof one or more miRNAs in the somatic transgenic animal.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 depicts a quantification of the number of AAV Cap genes detectedacross various tissues via RT-PCR based detection of RNA Cap sequencesor PCR based detection of DNA Cap sequences

FIG. 2 depicts a dendrogram from a hierarchical cluster analysis of AAVsbased on similarities of cap gene sequences. The dendrogram includes alarge cluster of AAV9 variants.

FIG. 3 is a schematic map of positions in AAV9 cap proteins of aminoacid variations of the AAV9 variants.

FIG. 4 depicts an amino acid comparison among immunological epitopes ofAAV cap protein. Sequences from Epitope 2 to AAV5 (357) correspond toSEQ ID NO: 186-190; sequences from AAV6 (367) to CLv-R7 (368) allcorrespond to SEQ ID NO: 187; sequence of CLg-F1 (368) corresponds toSEQ ID NO: 191; sequences from Epitope 3 to AAV5 (399) correspond to SEQID NO: 192-197; sequence of AAV6 (407) corresponds to SEQ ID NO: 194;sequences from AAV7 (408) to AAV9 (408) correspond to SEQ ID NO:198-200; sequences of Clv-R7 (408) and Clg-F1 (408) both correspond toSEQ ID NO: 200; sequence of CLg-F3 (408) corresponds to SEQ ID NO: 201;sequences from Epitope 4 to AAV5 (479) correspond to SEQ ID NO: 202 to207; sequence of AAV6 (493) corresponds to SEQ ID NO: 203; sequencesfrom AAV7 (495) to AAV9 (493) correspond to SEQ ID NO: 208-210;sequences from CLv-R7 (493) to CLv-R9 (493), CBr-E6 (492), and CSp-1(493) all correspond to SEQ ID NO: 210; sequence of CLg-F4 (493)corresponds to SEQ ID NO: 211; and sequence of CSp-3 (493) correspondsto SEQ ID NO: 212.

FIG. 5 depicts a dendrogram from a hierarchical cluster analysis of AAVsbased on similarities of cap gene sequences. The dendrogram includes acluster with two new AAV1 variants, which is expanded in the rightpanel, each having 4 amino acid differences from AAV1 cap protein.

FIG. 6 depicts a dendrogram from a hierarchical cluster analysis of AAVsbased on similarities of cap gene sequences. The dendrogram includes acluster of AAV1 variants, which is expanded in the right panel. Ckd-7and clv-3 have 4 amino acids different from AAV1 cap protein. Ckd-1,clv-4, ckd-4, ckd-6, and clv-12 have 3 amino acids different from AAV1cap protein.

FIG. 7 depicts a dendrogram from a hierarchical cluster analysis of AAVsbased on similarities of cap gene sequences. The dendrogram includes acluster of AAV6 variants, including Ckd-B6, CKd-B1, Ckd-B3, Ckd-B8,Ckd-B4, Ckd-B7, Ckd-H1, Ckd-H4, Ckd-H5, Ckd-H3 and Ckd-B2. Except forCkd-H3 and Ckd-B2 which have 3 a.a. different from AAV6 cap, all othershave 4 a.a. different from AAV6 cap.

FIG. 8 depicts a dendrogram from a hierarchical cluster analysis of AAVsbased on similarities of cap gene sequences. The dendrogram includes acluster of AAV6 variants, including Ckd-B6, CKd-B1, Ckd-B3, Ckd-B8,Ckd-B4, Ckd-B7, Ckd-H1, Ckd-H4, Ckd-H5. All of these new AAVs have 4a.a. different from AAV6 cap.

FIGS. 9A and B depict a dendrogram from a hierarchical cluster analysisof AAVs based on similarities of cap gene sequences. The most similarsequence in GenBank to CKd-H2 is AAV VRC-355. The latter was isolatedform simian adenovirus isolates in ATCC and is AAV6-like in terms ofidentity but has some different biological properties (J Virol, 2006,80:5082). CKd-H2 is similar to AAV1 and AAV6. When clustered with AAV1-9excluding AAV3, Ckd-H2 appears in a cluster with AAV6. (FIG. 9A.) Whenclustered with AAV1-9 including AAV3, Ckd-H2 appears in a cluster withAAV6. (FIG. 9B.)

FIG. 10 depicts sequence alignments among AAV1-AAV11 with forward (CapF)and reverse (AV2case) primers which are used together for RT-PCR basedrecovery of AAV cap coding sequences. Sequences from capF (1) toAAV2(2163) correspond to SEQ ID NO: 213-215; sequences of AAV3(2169),AAV6(2168), and AAV8(2081) to AAV11(1846) all correspond to SEQ ID NO:214; sequence of AAV4(2220) corresponds to SEQ ID NO: 216; sequence ofAAV7(2182) corresponds to SEQ ID NO: 217; sequences of AAV2cas (1) toAAV8(4362) correspond to SEQ ID NO: 218-225; and sequence of AAV9(4355)corresponds to SEQ ID NO: 220.

FIG. 11 depicts sequence alignments among various AAVs with threeforward primers which are used together for RT-PCR based recovery of AAVcap coding sequences. Sequences from capF22 to AAV5(1856) correspond toSEQ ID NO: 226-231; sequences of AAV6(1836) to AAV9(1744) all correspondto SEQ ID NO: 227; sequence of AAV-rh39(1850) to AAV5(1894) correspondsto SEQ ID NO: 232-238; sequence of AAV6(1874), AAV8(1791) and AAV9(1782)all correspond to SEQ ID NO: 234; sequence of AAV7(1888) corresponds toSEQ ID NO: 239; sequences of capF201 (1) to AAV8(1928) correspond to SEQID NO: 240-248; sequence of AAV9(1919) corresponds to SEQ ID NO: 248;and sequence of AAV-rh39(2022) corresponds to SEQ ID NO: 249.

FIG. 12A depicts transduction efficiency of AAV9 and Csp-3 vectors indifferent organs. FIG. 12B depicts transduction efficient of AAVvariants in skeletal muscle.

FIG. 13 depicts transduction efficiency of AAV variants in skeletalmuscle at different doses.

FIG. 14 depicts transduction efficiency of AAV variants by differentroutes of administration.

FIG. 15 depicts transduction efficiency of AAV variants in Lung.

DETAILED DESCRIPTION

Adeno-associated virus (AAV) is a small (˜26 nm) replication-defective,nonenveloped virus, that depends on the presence of a second virus, suchas adenovirus or herpes virus, for its growth in cells. AAV is not knownto cause disease and induces a very mild immune response. AAV can infectboth dividing and non-dividing cells and may incorporate its genome intothat of the host cell. These features make AAV a very attractivecandidate for creating viral vectors for gene therapy. Prototypical AAVvectors based on serotype 2 provided a proof-of-concept for non-toxicand stable gene transfer in murine and large animal models, butexhibited poor gene transfer efficiency in many major target tissues.The invention in some aspects seeks to overcome this shortcoming byproviding novel AAVs having distinct tissue targeting capabilities forgene therapy and research applications.

In some aspects of the invention new AAV capsid proteins are providedthat have distinct tissue targeting capabilities. In some embodiments,an AAV capsid protein is isolated from the tissue to which an AAVcomprising the capsid protein targets. In some aspects, methods fordelivering a transgene to a target tissue in a subject are provided. Thetransgene delivery methods may be used for gene therapy (e.g., to treatdisease) or research (e.g., to create a somatic transgenic animal model)applications.

Methods for Discovering AAVs

Much of the biology of AAV is dictated by its capsid. Consequently,methods for discovering novel AAVs have been largely focused onisolating DNA sequences for AAV capsids. A central feature of theadeno-associated virus (AAV) latent life cycle is persistence in theform of integrated and/or episomal genomes in a host cell. To date, theprimary methods used for isolating novel AAV include PCR based molecularrescue of latent AAV DNA genomes, infectious virus rescue of latentproviral genome from tissue DNAs in vitro in the presence of adenovirushelper function, and rescue of circular proviral genome from tissue DNAsby rolling-circle-linear amplification, mediated by an isothermal phagePhi-29 polymerase. All of these isolation methods take advantage of thelatency of AAV proviral DNA genomes and focus on rescuing persistentviral genomic DNA. A major challenge in DNA-targeted AAV isolation isthat the abundance of persisted AAV genomes is often very low in mosttissues particularly in human tissues, which makes AAV rescueunachievable in many cases. In some cases, PCR-based DNA recoverymethods capture all endogenous AAVs, tinting the libraries of AAVproviral sequences with singleton bearing nonfunctional species.

Endogenous latent AAV genomes are transcriptionally active in mammaliancells (e.g., cells of nonhuman primate tissues such as liver, spleen andlymph nodes). Without wishing to bound by theory, it is hypothesizedthat to maintain AAV persistence in host, low levels of transcriptionfrom AAV genes could be required and the resulting cap RNA could serveas more suitable and abundant substrates to retrieve functional capsequences for vector development. Both rep and cap gene transcripts aredetected with variable abundances by RNA detection methods (e.g.,RT-PCR). The presence of cap gene transcripts and ability to generatecDNA of cap RNA through reverse transcription (RT) in vitrosignificantly increases abundance of templates for PCR-based rescue ofnovel cap sequences from tissues and enhance the sensitivity of novelAAV discovery.

Novel cap sequences may also be identified by transfecting cells withtotal cellular DNAs isolated from the tissues that harbor proviral AAVgenomes at very low abundance, The cells may be further transfected withgenes that provide helper virus function (e.g., adenovirus) to triggerand/or boost AAV gene transcription in the transfected cells. Novel capsequences of the invention may be identified by isolating cap mRNA fromthe transfected cells, creating cDNA from the mRNA (e.g., by RT-PCR) andsequencing the cDNA.

Isolated Capsid Proteins and Nucleic Acids Encoding the Sames

AAVs are natural inhabitants in mammals. AAVs isolated from mammals,particularly non-human primates, are useful for creating gene transfervectors for clinical development and human gene therapy applications.The invention provides in some aspects novel AAVs that have beendiscovered in various non-human primate tissues using the methodsdisclosed herein. Nucleic acids encoding capsid proteins of these novelAAVs have been discovered in both viral genomic DNA and mRNA isolatedfrom the non-human primate tissues. Nucleic acid and protein sequencesas well as other information regarding the AAVs are set forth in Tables3A-C and in the sequence listing.

Isolated nucleic acids of the invention that encode AAV capsid proteinsinclude any nucleic acid having a sequence as set forth in any one ofSEQ ID NOs 13-86 as well as any nucleic acid having a sequence withsubstantial homology thereto. In some embodiments, the inventionprovides an isolated nucleic acid that has substantial homology with anucleic acid having a sequence as set forth in any one of SEQ ID NOs13-86, but that does not encode a protein having an amino acid sequenceas set forth in any one of SEQ ID NOs 177-183.

Furthermore, isolated AAV capsid proteins of the invention include anyprotein having an amino acid sequence as set forth in any one of SEQ IDNOs 87-160 and 171-176, as well as any protein having substantialhomology thereto. In some embodiments, the invention provides anisolated capsid protein that has substantial homology with a proteinhaving a sequence as set forth in any one of SEQ ID NOs 87-160 and171-176, but that does not have an amino acid sequence as set forth inany one of SEQ ID NOs 177-183.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. The term “substantial homology”, whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in about 90 to 100% of the alignedsequences. When referring to a polypeptide, or fragment thereof, theterm “substantial homology” indicates that, when optimally aligned withappropriate gaps, insertions or deletions with another polypeptide,there is nucleotide sequence identity in about 90 to 100% of the alignedsequences. The term “highly conserved” means at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. In some cases, highly conserved may refer to 100% identity.Identity is readily determined by one of skill in the art by, forexample, the use of algorithms and computer programs known by those ofskill in the art.

As described herein, alignments between sequences of nucleic acids orpolypeptides are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs, such as“Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI utilities may also be used. There are also anumber of algorithms known in the art which can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using BLASTN, which provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences. Similar programs are available for the comparison of aminoacid sequences, e.g., the “Clustal X” program, BLASTP. Typically, any ofthese programs are used at default settings, although one of skill inthe art can alter these settings as needed. Alternatively, one of skillin the art can utilize another algorithm or computer program whichprovides at least the level of identity or alignment as that provided bythe referenced algorithms and programs. Alignments may be used toidentify corresponding amino acids between two proteins or peptides. A“corresponding amino acid” is an amino acid of a protein or peptidesequence that has been aligned with an amino acid of another protein orpeptide sequence. Corresponding amino acids may be identical ornon-identical. A corresponding amino acid that is a non-identical aminoacid may be referred to as a variant amino acid. Tables of correspondingamino acids among various AAV variants is provided in Table 4A-C, forexample.

Alternatively for nucleic acids, homology can be determined byhybridization of polynucleotides under conditions which form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. DNA sequences that are substantially homologous canbe identified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.

A “nucleic acid” sequence refers to a DNA or RNA sequence. In someembodiments, the term nucleic acid captures sequences that include anyof the known base analogues of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

Proteins and nucleic acids of the invention are isolated. As usedherein, the term “isolated” means removed from a natural environment orartificially produced. As used herein with respect to nucleic acids, theterm “isolated” means: (i) amplified in vitro by, for example,polymerase chain reaction (PCR); (ii) recombinantly produced by cloning;(iii) purified, as by cleavage and gel separation; or (iv) synthesizedby, for example, chemical synthesis. An isolated nucleic acid is onewhich is readily manipulable by recombinant DNA techniques well known inthe art. Thus, a nucleotide sequence contained in a vector in which 5′and 3′ restriction sites are known or for which polymerase chainreaction (PCR) primer sequences have been disclosed is consideredisolated but a nucleic acid sequence existing in its native state in itsnatural host is not. An isolated nucleic acid may be substantiallypurified, but need not be. For example, a nucleic acid that is isolatedwithin a cloning or expression vector is not pure in that it maycomprise only a tiny percentage of the material in the cell in which itresides. Such a nucleic acid is isolated, however, as the term is usedherein because it is readily manipulable by standard techniques known tothose of ordinary skill in the art. As used herein with respect toproteins or peptides, the term “isolated” refers to a protein or peptidethat has been isolated from its natural environment or artificiallyproduced (e.g., by chemical synthesis, by recombinant DNA technology,etc.).

The skilled artisan will also realize that conservative amino acidsubstitutions may be made to provide functionally equivalent variants,or homologs of the capsid proteins. In some aspects the inventionembraces sequence alterations that result in conservative amino acidsubstitutions. As used herein, a conservative amino acid substitutionrefers to an amino acid substitution that does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references that compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. Conservative substitutionsof amino acids include substitutions made among amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D. Therefore, one can make conservativeamino acid substitutions to the amino acid sequence of the proteins andpolypeptides disclosed herein.

An example of an isolated nucleic acid that encodes an AAV capsidprotein is a nucleic acid having a sequence selected from the groupconsisting of: SEQ ID NO: 13-86. A fragment of an isolated nucleic acidencoding a AAV capsid sequence may be useful for constructing a nucleicacid encoding a desired capsid sequence. Fragments may be of anyappropriate length. The fragment may be a fragment that does not encodea peptide that is identical to a sequence of any one of SEQ ID NOs:179-185. For example, a fragment of nucleic acid sequence encoding avariant amino acid (compared with a known AAV serotype) may be used toconstruct, or may be incorporated within, a nucleic acid sequenceencoding an AAV capsid sequence to alter the properties of the AAVcapsid. For example, a nucleic sequence encoding an AAV variant (e.g.,Csp3) may comprise n amino acid variants compared with a known AAVserotype (e.g., AAV9). A recombinant cap sequence may be constructedhaving one or more of the n amino acid variants by incorporatingfragments of a nucleic acid sequence comprising a region encoding avariant amino acid into the sequence of a nucleic acid encoding theknown AAV serotype. The fragments may be incorporated by any appropriatemethod, including using site directed mutagenesis. Thus, new AAVvariants may be created having new properties.

Recombinant AAVs

In some aspects, the invention provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenisolated from its natural environment (e.g., from a host cell, tissue,or subject) or artificially produced. Isolated AAVs may be producedusing recombinant methods. Such AAVs are referred to herein as“recombinant AAVs”. Recombinant AAVs (rAAVs) preferably havetissue-specific targeting capabilities, such that a transgene of therAAV will be delivered specifically to one or more predeterminedtissue(s). The AAV capsid is an important element in determining thesetissue-specific targeting capabilities. Thus, an rAAV having a capsidappropriate for the tissue being targeted can be selected. In someembodiments, the rAAV comprises a capsid protein having an amino acidsequence as set forth in any one of SEQ ID NOs 87-160 and 171-178, or aprotein having substantial homology thereto.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art. (See, for example, US 2003/0138772), thecontents of which are incorporated herein by reference in theirentirety). Typically the methods involve culturing a host cell whichcontains a nucleic acid sequence encoding an AAV capsid protein (e.g., anucleic acid having a sequence as set forth in any one of SEQ ID NOs13-86) or fragment thereof; a functional rep gene; a recombinant AAVvector composed of, AAV inverted terminal repeats (ITRs) and atransgene; and sufficient helper functions to permit packaging of therecombinant AAV vector into the AAV capsid proteins.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this invention are known to those with skillin nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virionsare well known and the selection of a suitable method is not alimitation on the present invention. See, e.g., K. Fisher et al, J.Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (described in detail in U.S. Pat. No. 6,001,650).Typically, the recombinant AAVs are produced by transfecting a host cellwith an recombinant AAV vector (comprising a transgene) to be packagedinto AAV particles, an AAV helper function vector, and an accessoryfunction vector. An AAV helper function vector encodes the “AAV helperfunction” sequences (i.e., rep and cap), which function in trans forproductive AAV replication and encapsidation. Preferably, the AAV helperfunction vector supports efficient AAV vector production withoutgenerating any detectable wild-type AAV virions (i.e., AAV virionscontaining functional rep and cap genes). Non-limiting examples ofvectors suitable for use with the present invention include pHLP19,described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described inU.S. Pat. No. 6,156,303, the entirety of both incorporated by referenceherein. The accessory function vector encodes nucleotide sequences fornon-AAV derived viral and/or cellular functions upon which AAV isdependent for replication (i.e., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the invention provides transfected host cells. The term“transfection” is used to refer to the uptake of foreign DNA by a cell,and a cell has been “transfected” when exogenous DNA has been introducedinside the cell membrane. A number of transfection techniques aregenerally known in the art. See, e.g., Graham et al. (1973) Virology,52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual,Cold Spring Harbor Laboratories, New York, Davis et al. (1986) BasicMethods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into whichan exogenous DNA segment, such as DNA segment that leads to thetranscription of a biologically-active polypeptide or production of abiologically active nucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA, miRNA inhibitor) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the invention are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

“Recombinant AAV (rAAV) vectors” of the invention are typically composedof, at a minimum, a transgene and its regulatory sequences, and 5′ and3′ AAV inverted terminal repeats (ITRs). It is this recombinant AAVvector which is packaged into a capsid protein and delivered to aselected target cell. In some embodiments, the transgene is a nucleicacid sequence, heterologous to the vector sequences, which encodes apolypeptide, protein, functional RNA molecule (e.g., miRNA, miRNAinhibitor) or other gene product, of interest. The nucleic acid codingsequence is operatively linked to regulatory components in a mannerwhich permits transgene transcription, translation, and/or expression ina cell of a target tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present invention is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elementsnecessary which are operably linked to the transgene in a manner whichpermits its transcription, translation and/or expression in a celltransfected with the plasmid vector or infected with the virus producedby the invention. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be “operably” linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present inventionmay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. Another vector element that may be used is an internalribosome entry site (IRES). An IRES sequence is used to produce morethan one polypeptide from a single gene transcript. An IRES sequencewould be used to produce a protein that contain more than onepolypeptide chains. Selection of these and other common vector elementsare conventional and many such sequences are available [see, e.g.,Sambrook et al, and references cited therein at, for example, pages 3.183.26 and 16.17 16.27 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1989]. In some embodiments, a Footand Mouth Disease Virus 2A sequence is included in polyprotein; this isa small peptide (approximately 18 amino acids in length) that has beenshown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO,1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin,C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity ofthe 2A sequence has previously been demonstrated in artificial systemsincluding plasmids and gene therapy vectors (AAV and retroviruses)(Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., JVirology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy,2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe,P et al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al.,Gene Therapy, 2001; 8: 811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of theinvention may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al, J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. Exemplary tissue-specific regulatory sequences include, butare not limited to the following tissue specific promoters: aliver-specific thyroxin binding globulin (TBG) promoter, a insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatinekinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosinheavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.Other exemplary promoters include Beta-actin promoter, hepatitis B viruscore promoter, Sandig et al., Gene Ther., 3:1002-9 (1996);alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther.,7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol.Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J.Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cellreceptor α-chain promoter, neuronal such as neuron-specific enolase(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15(1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc.Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgfgene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among otherswhich will be apparent to the skilled artisan.

In some embodiments, one or more bindings sites for one or more ofmiRNAs are incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of an subjectharboring the transgene. The skilled artisan will appreciate thatbinding sites may be selected to control the expression of a trangene ina tissue specific manner. For example, binding sites for theliver-specific miR-122 may be incorporated into a transgene to inhibitexpression of that transgene in the liver. The target sites in the mRNAmay be in the 5′ UTR, the 3′ UTR or in the coding region. Typically, thetarget site is in the 3′ UTR of the mRNA. Furthermore, the transgene maybe designed such that multiple miRNAs regulate the mRNA by recognizingthe same or multiple sites. The presence of multiple miRNA binding sitesmay result in the cooperative action of multiple RISCs and providehighly efficient inhibition of expression. The target site sequence maycomprise a total of 5-100, 10-60, or more nucleotides. The target sitesequence may comprise at least 5 nucleotides of the sequence of a targetgene binding site.

Recombinant AAV Vector: Transgene Coding Sequences

The composition of the transgene sequence of the rAAV vector will dependupon the use to which the resulting vector will be put. For example, onetype of transgene sequence includes a reporter sequence, which uponexpression produces a detectable signal. In another example, thetrangene encodes a therapeutic protein or therapeutic functional RNA. Inanother example, the transgene encodes a protein or functional RNA thatis intended to be used for research purposes, e.g., to create a somatictransgenic animal model harboring the transgene, e.g., to study thefunction of the transgene product. In another example, the transgeneencodes a protein or functional RNA that is intended to be used tocreate an animal model of disease. Appropriate transgene codingsequences will be apparent to the skilled artisan.

Reporter sequences that may be provided in a transgene include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art. When associated with regulatory elements which drivetheir expression, the reporter sequences, provide signals detectable byconventional means, including enzymatic, radiographic, colorimetric,fluorescence or other spectrographic assays, fluorescent activating cellsorting assays and immunological assays, including enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for β-galactosidase activity. Where the transgene is greenfluorescent protein or luciferase, the vector carrying the signal may bemeasured visually by color or light production in a luminometer. Suchreporters can, for example, be useful in verifying the tissue-specifictargeting capabilities and tissue specific promoter regulatory activityof an rAAV.

In some aspects, the invention provides rAAV vectors for use in methodsof preventing or treating one or more genetic deficiencies ordysfunctions in a mammal, such as for example, a polypeptide deficiencyor polypeptide excess in a mammal, and particularly for treating orreducing the severity or extent of deficiency in a human manifesting oneor more of the disorders linked to a deficiency in such polypeptides incells and tissues. The method involves administration of an rAAV vectorthat encodes one or more therapeutic peptides, polypeptides, siRNAs,microRNAs, antisense nucleotides, etc. in a pharmaceutically-acceptablecarrier to the subject in an amount and for a period of time sufficientto treat the deficiency or disorder in the subject suffering from such adisorder.

Thus, the invention embraces the delivery of rAAV vectors encoding oneor more peptides, polypeptides, or proteins, which are useful for thetreatment or prevention of disease states in a mammalian subject.Exemplary therapeutic proteins include one or more polypeptides selectedfrom the group consisting of growth factors, interleukins, interferons,anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosisagents, coagulation factors, anti-tumor factors. Other non-limitingexamples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF,G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF,VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18.

The rAAV vectors may comprise a gene to be transferred to a subject totreat a disease associated with reduced expression, lack of expressionor dysfunction of the gene. Exemplary genes and associated diseasestates include, but are not limited to: glucose-6-phosphatase,associated with glycogen storage deficiency type 1A;phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency;galactose-1 phosphate uridyl transferase, associated with galactosemia;phenylalanine hydroxylase, associated with phenylketonuria; branchedchain alpha-ketoacid dehydrogenase, associated with Maple syrup urinedisease; fumarylacetoacetate hydrolase, associated with tyrosinemia type1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia;medium chain acyl CoA dehydrogenase, associated with medium chain acetylCoA deficiency; omithine transcarbamylase, associated with omithinetranscarbamylase deficiency; argininosuccinic acid synthetase,associated with citrullinemia; low density lipoprotein receptor protein,associated with familial hypercholesterolemia;UDP-glucouronosyltransferase, associated with Crigler-Najjar disease;adenosine deaminase, associated with severe combined immunodeficiencydisease; hypoxanthine guanine phosphoribosyl transferase, associatedwith Gout and Lesch-Nyan syndrome; biotinidase, associated withbiotinidase deficiency; beta-glucocerebrosidase, associated with Gaucherdisease; beta-glucuronidase, associated with Sly syndrome; peroxisomemembrane protein 70 kDa, associated with Zellweger syndrome;porphobilinogen deaminase, associated with acute intermittent porphyria;alpha-1 antitrypsin for treatment of alpha-1 antitrypsin deficiency(emphysema); erythropoietin for treatment of anemia due to thalassemiaor to renal failure; vascular endothelial growth factor, angiopoietin-1,and fibroblast growth factor for the treatment of ischemic diseases;thrombomodulin and tissue factor pathway inhibitor for the treatment ofoccluded blood vessels as seen in, for example, atherosclerosis,thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), andtyrosine hydroxylase (TH) for the treatment of Parkinson's disease; thebeta adrenergic receptor, anti-sense to, or a mutant form of,phospholamban, the sarco(endo)plasmic reticulum adenosinetriphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for thetreatment of congestive heart failure; a tumor suppessor gene such asp53 for the treatment of various cancers; a cytokine such as one of thevarious interleukins for the treatment of inflammatory and immunedisorders and cancers; dystrophin or minidystrophin and utrophin orminiutrophin for the treatment of muscular dystrophies; and, insulin forthe treatment of diabetes.

The rAAVs of the invention can be used to restore the expression ofgenes that are reduced in expression, silenced, or otherwisedysfunctional in a subject (e.g., a tumor suppressor that has beensilenced in a subject having cancer). The rAAVs of the invention canalso be used to knockdown the expression of genes that are aberrantlyexpressed in a subject (e.g., an oncogene that is expressed in a subjecthaving cancer). In some embodiments, an rAAV vector comprising a nucleicacid encoding a gene product associated with cancer (e.g., tumorsuppressors) may be used to treat the cancer, by administering a rAAVharboring the rAAV vector to a subject having the cancer. In someembodiments, an rAAV vector comprising a nucleic acid encoding a smallinterfering nucleic acid (e.g., shRNAs, miRNAs) that inhibits theexpression of a gene product associated with cancer (e.g., oncogenes)may be used to treat the cancer, by administering a rAAV harboring therAAV vector to a subject having the cancer. In some embodiments, an rAAVvector comprising a nucleic acid encoding a gene product associated withcancer (or a functional RNA that inhibits the expression of a geneassociated with cancer) may be used for research purposes, e.g., tostudy the cancer or to identify therapeutics that treat the cancer. Thefollowing is a non-limiting list of exemplary genes known to beassociated with the development of cancer (e.g., oncogenes and tumorsuppressors): AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1,ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5,ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2,BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1,CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44,CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9,CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1,CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1,COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1,CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8,DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2,ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES,FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB,FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2,GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB,HSPA4, HSPA5, HSPA8, HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA,IER3, IFITM1, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1,IRF3, ITGA3, ITGA6, ITGB4, JAK1, JARID1A, JUN, JUNB, JUND, K-ALPHA-1,KIT, KITLG, KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK,LCN2, LEP, LITAF, LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2, MAP3K8,MAPK12, MAPK13, MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2,MDM4, MET, MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA,MSH2, MSH6, MT3, MYB, MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK,NEO1, NF1, NF2, NFKB1, NFKB2, NFSF7, NID, NINJ1, NMBR, NME1, NME2, NME3,NOTCH1, NOTCH2, NOTCH4, NPM1, NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1,NSEP1, OSM, PA2G4, PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB,PDGFRA, PDPK1, PEA15, PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB, PIK3CG,PIM1, PKM2, PKMYT1, PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2,PRDX4, PRKAR1A, PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA,PTN, PTPRN, RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RARA, RARB,RASGRF1, RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA,RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6KB1, RRM1, SARS, SELENBP1, SEMA3C,SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVATP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, SMPD1, SNAI2, SND1,SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1,STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C,TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1,TIMP3, TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B,TNFRSF6, TNFSF7, TNK1, TOB1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1,TRADD, TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1,USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1,XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9.

A rAAV vector may comprise as a transgene, a nucleic acid encoding aprotein or functional RNA that modulates apoptosis. The following is anon-limiting list of genes associated with apoptosis and nucleic acidsencoding the products of these genes and their homologues and encodingsmall interfering nucleic acids (e.g., shRNAs, miRNAs) that inhibit theexpression of these genes and their homologues are useful as transgenesin certain embodiments of the invention: RPS27A, ABL1, AKT1, APAF1, BAD,BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10,BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2,BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L,BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARDS,CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7,CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB,FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, MCL1, NOL3, PYCARD, RIPK1,RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B,TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25,CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD40LG,FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1,TRAF2, TRAF3, TRAF4, and TRAF5.

The skilled artisan will also realize that in the case of transgenesencoding proteins or polypeptides, that mutations that results inconservative amino acid substitutions may be made in a transgene toprovide functionally equivalent variants, or homologs of a protein orpolypeptide. In some aspects the invention embraces sequence alterationsthat result in conservative amino acid substitution of a transgene. Insome embodiments, the transgene comprises a gene having a dominantnegative mutation. For example, a transgene may express a mutant proteinthat interacts with the same elements as a wild-type protein, andthereby blocks some aspect of the function of the wild-type protein.

Useful transgene products also include miRNAs. miRNAs and other smallinterfering nucleic acids regulate gene expression via target RNAtranscript cleavage/degradation or translational repression of thetarget messenger RNA (mRNA). miRNAs are natively expressed, typically asfinal 19-25 non-translated RNA products. miRNAs exhibit their activitythrough sequence-specific interactions with the 3′ untranslated regions(UTR) of target mRNAs. These endogenously expressed miRNAs form hairpinprecursors which are subsequently processed into a miRNA duplex, andfurther into a “mature” single stranded miRNA molecule. This maturemiRNA guides a multiprotein complex, miRISC, which identifies targetsite, e.g., in the 3′ UTR regions, of target mRNAs based upon theircomplementarity to the mature miRNA.

The following non-limiting list of miRNA genes, and their homologues,are useful as transgenes or as targets for small interfering nucleicacids encoded by transgenes (e.g., miRNA sponges, antisenseoligonucleotides, TuD RNAs) in certain embodiments of the methods:hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c,hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*,hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*,hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*,hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*,hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107,hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178,hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183,hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201,hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206,hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122,hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p,hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227,hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233,hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124,hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246,hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251,hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b,hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p,hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*,hsa-miR-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262,hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267,hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272,hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b,hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278,hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282,hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287,hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291,hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295,hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299,hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304,hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a,hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*,hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a,hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b,hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138,hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p,hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144,hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*,hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b,hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR-149,hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p,hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155,hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*,hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*,hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b,hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*,hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*,hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*,hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a,hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b,hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p,hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*,hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b,hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p,hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1*,hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b,hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*,hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a,hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*,hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212,hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b,hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*,hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22,hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221,hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*,hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*,hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*,hsa-miR-26a, hsa-miR-26a-1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*,hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p,hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298,hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b,hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300,hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b,hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*,hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30b,hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d,hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*,hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c,hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p,hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329,hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p,hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p,hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p,hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340,hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346,hsa-miR-34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p,hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p,hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367,hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370,hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*,hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375,hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377,hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*,hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383,hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411,hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p,hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*,hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*,hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a,hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452,hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p,hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484,hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p,hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489,hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p,hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495,hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p,hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p,hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503,hsa-miR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507,hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p,hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p,hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b,hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p,hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*,hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a-3p,hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR-518c*,hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e, hsa-miR-518e*,hsa-miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p,hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*,hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR-520c-3p,hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f,hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR-523,hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p,hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-532-5p,hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p,hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p,hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-548b-5p, hsa-miR-548c-3p,hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548e,hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j,hsa-miR-548k, hsa-miR-548l, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o,hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a,hsa-miR-551b, hsa-miR-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554,hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558,hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564,hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570,hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p,hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578,hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p,hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587,hsa-miR-588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p,hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595,hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p,hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618,hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623,hsa-miR-624, hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626,hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*,hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634,hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639,hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644,hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649,hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p,hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658,hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663,hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668,hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708,hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744,hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766,hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p,hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-770-5p, hsa-miR-802,hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p,hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*,hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p,hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890,hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9,hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923,hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b,hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934,hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939,hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944,hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a,hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*.

A miRNA inhibits the function of the mRNAs it targets and, as a result,inhibits expression of the polypeptides encoded by the mRNAs. Thus,blocking (partially or totally) the activity of the miRNA (e.g.,silencing the miRNA) can effectively induce, or restore, expression of apolypeptide whose expression is inhibited (derepress the polypeptide).In one embodiment, derepression of polypeptides encoded by mRNA targetsof a miRNA is accomplished by inhibiting the miRNA activity in cellsthrough any one of a variety of methods. For example, blocking theactivity of a miRNA can be accomplished by hybridization with a smallinterfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge,TuD RNA) that is complementary, or substantially complementary to, themiRNA, thereby blocking interaction of the miRNA with its target mRNA.As used herein, an small interfering nucleic acid that is substantiallycomplementary to a miRNA is one that is capable of hybridizing with amiRNA, and blocking the miRNA's activity. In some embodiments, an smallinterfering nucleic acid that is substantially complementary to a miRNAis an small interfering nucleic acid that is complementary with themiRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 bases. In some embodiments, an small interfering nucleic acidsequence that is substantially complementary to a miRNA, is an smallinterfering nucleic acid sequence that is complementary with the miRNAat, at least, one base.

A “miRNA Inhibitor” is an agent that blocks miRNA function, expressionand/or processing. For instance, these molecules include but are notlimited to microRNA specific antisense, microRNA sponges, tough decoyRNAs (TuD RNAs) and microRNA oligonucleotides (double-stranded, hairpin,short oligonucleotides) that inhibit miRNA interaction with a Droshacomplex. MicroRNA inhibitors can be expressed in cells from a transgenesof a rAAV vector, as discussed above. MicroRNA sponges specificallyinhibit miRNAs through a complementary heptameric seed sequence (Ebert,M. S. Nature Methods, Epub Aug. 12, 2007). In some embodiments, anentire family of miRNAs can be silenced using a single sponge sequence.TuD RNAs achieve efficient and long-term-suppression of specific miRNAsin mammalian cells (See, e.g., Takeshi Haraguchi, et al., Nucleic AcidsResearch, 2009, Vol. 37, No. 6 e43, the contents of which relating toTuD RNAs are incorporated herein by reference). Other methods forsilencing miRNA function (derepression of miRNA targets) in cells willbe apparent to one of ordinary skill in the art.

In some embodiments, the cloning capacity of the recombinant RNA vectormay limited and a desired coding sequence may require the completereplacement of the virus's 4.8 kilobase genome. Large genes may,therefore, not be suitable for use in a standard recombinant AAV vector,in some cases. The skilled artisan will appreciate that options areavailable in the art for overcoming a limited coding capacity. Forexample, the AAV ITRs of two genomes can anneal to form head to tailconcatamers, almost doubling the capacity of the vector. Insertion ofsplice sites allows for the removal of the ITRs from the transcript.Other options for overcoming a limited cloning capacity will be apparentto the skilled artisan.

Somatic Transgenic Animal Models Produced Using rAAV-Based Gene Transfer

The invention also involves the production of somatic transgenic animalmodels of disease using recombinant Adeno-Associated Virus (rAAV) basedmethods. The methods are based, at least in part, on the observationthat AAV serotypes and variants thereof mediate efficient and stablegene transfer in a tissue specific manner in adult animals. The rAAVelements (capsid, promoter, transgene products) are combined to achievesomatic transgenic animal models that express a stable transgene in atime and tissue specific manner. The somatic transgenic animal producedby the methods of the invention can serve as useful models of humandisease, pathological state, and/or to characterize the effects of genefor which the function (e.g., tissue specific, disease role) is unknownor not fully understood. For example, an animal (e.g., mouse) can beinfected at a distinct developmental stage (e.g., age) with a rAAVcomprising a capsid having a specific tissue targeting capability (e.g.,liver, heart, pancreas) and a transgene having a tissue specificpromoter driving expression of a gene involved in disease. Uponinfection, the rAAV infects distinct cells of the target tissue andproduces the product of the transgene.

In some embodiments, the sequence of the coding region of a transgene ismodified. The modification may alter the function of the product encodedby the transgene. The effect of the modification can then be studied invivo by generating a somatic transgenic animal model using the methodsdisclosed herein. In some embodiments, modification of the sequence ofcoding region is a nonsense mutation that results in a fragment (e.g., atruncated version). In other cases, the modification is a mis sensemutation that results in an amino acid substitution. Other modificationsare possible and will be apparent to the skilled artisan.

In some embodiments, the transgene causes a pathological state. Atransgene that causes a pathological state is a gene whose product has arole in a disease or disorder (e.g., causes the disease or disorder,makes the animal susceptible to the disease or disorder) and/or mayinduce the disease or disorder in the animal. The animal can then beobserved to evaluate any number of aspects of the disease (e.g.,progression, response to treatment, etc). These examples are not meantto be limiting, other aspects and examples are disclosed herein anddescribed in more detail below.

The invention in some aspects, provide methods for producing somatictransgenic animal models through the targeted destruction of specificcell types. For example, models of type 1 diabetes can be produced bythe targeted destruction of pancreatic Beta-islets. In other examples,the targeted destruction of specific cell types can be used to evaluatethe role of specific cell types on human disease. In this regard,transgenes that encode cellular toxins (e.g., diphtheria toxin A (DTA))or pro-apoptotic genes (NTR, Box, etc.) can be useful as transgenes forfunctional ablation of specific cell types. Other exemplary transgenes,whose products kill cells are embraced by the methods disclosed hereinand will be apparent to one of ordinary skill in the art.

The invention in some aspects, provides methods for producing somatictransgenic animal models to study the long-term effects ofover-expression or knockdown of genes. The long term over expression orknockdown (e.g., by shRNA, miRNA, miRNA inhibitor, etc.) of genes inspecific target tissues can disturb normal metabolic balance andestablish a pathological state, thereby producing an animal model of adisease, such as, for example, cancer. The invention in some aspects,provides methods for producing somatic transgenic animal models to studythe long-term effects of over-expression or knockdown of gene ofpotential oncogenes and other genes to study tumorigenesis and genefunction in the targeted tissues. Useful transgene products includeproteins that are known to be associated with cancer and smallinterfering nucleic acids inhibiting the expression of such proteins.Other suitable transgenes may be readily selected by one of skill in theart provided that they are useful for creating animal models oftissue-specific pathological state and/or disease.

Recombinant AAV Administration Methods

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, preferably suspended ina physiologically compatible carrier (i.e., in a composition), may beadministered to a subject, i.e. host animal, such as a human, mouse,rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig,hamster, chicken, turkey, or a non-human primate (e.g, Macaque). In someembodiments a host animal does not include a human.

Delivery of the rAAVs to a mammalian subject may be by, for example,intramuscular injection or by administration into the bloodstream of themammalian subject. Administration into the bloodstream may be byinjection into a vein, an artery, or any other vascular conduit. In someembodiments, the rAAVs are administered into the bloodstream by way ofisolated limb perfusion, a technique well known in the surgical arts,the method essentially enabling the artisan to isolate a limb from thesystemic circulation prior to administration of the rAAV virions. Avariant of the isolated limb perfusion technique, described in U.S. Pat.No. 6,177,403, can also be employed by the skilled artisan to administerthe virions into the vasculature of an isolated limb to potentiallyenhance transduction into muscle cells or tissue. Moreover, in certaininstances, it may be desirable to deliver the virions to the CNS of asubject. By “CNS” is meant all cells and tissue of the brain and spinalcord of a vertebrate. Thus, the term includes, but is not limited to,neuronal cells, glial cells, astrocytes, cereobrospinal fluid (CSF),interstitial spaces, bone, cartilage and the like. Recombinant AAVs maybe delivered directly to the CNS or brain by injection into, e.g., theventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), spinal cord and neuromuscularjunction, or cerebellar lobule, with a needle, catheter or relateddevice, using neurosurgical techniques known in the art, such as bystereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429,1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.11:2315-2329, 2000).

The compositions of the invention may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In some embodiments, acomposition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more differentrAAVs each having one or more different transgenes.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. The selection of the carrier is not a limitation of the presentinvention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The rAAVS are administered in sufficient amounts to transfect the cellsof a desired tissue and to provide sufficient levels of gene transferand expression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, intratumoral, and other parental routes ofadministration. Routes of administration may be combined, if desired.

The dose of rAAV virions required to achieve a particular “therapeuticeffect,” e.g., the units of dose in genome copies/per kilogram of bodyweight (GC/kg), will vary based on several factors including, but notlimited to: the route of rAAV virion administration, the level of geneor RNA expression required to achieve a therapeutic effect, the specificdisease or disorder being treated, and the stability of the gene or RNAproduct. One of skill in the art can readily determine a rAAV viriondose range to treat a patient having a particular disease or disorderbased on the aforementioned factors, as well as other factors that arewell known in the art.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of an rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, a effective amount of the rAAV is generally in the range offrom about 1 ml to about 100 ml of solution containing from about 10⁹ to10¹⁶ genome copies. In some cases, a dosage between about 10¹¹ to 10¹²rAAV genome copies is appropriate. In certain embodiments, 10¹² rAAVgenome copies is effective to target heart, liver, and pancreas tissues.In some cases, stable transgenic animals are produced by multiple dosesof an rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy(2005) 12, 171-178, the contents of which are incorporated herein byreference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the rAAV-basedtherapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, or orally, intraperitoneally, or byinhalation. In some embodiments, the administration modalities asdescribed in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (eachspecifically incorporated herein by reference in its entirety) may beused to deliver rAAVs. In some embodiments, a preferred mode ofadministration is by portal vein injection.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present invention intosuitable host cells. In particular, the rAAV vector delivered trangenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trails examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (ie., ultrasound) has beenused and described in U.S. Pat. No. 5,656,016 as a device for enhancingthe rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Kits and Related Compositions

The agents described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise proces sable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for animal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to ananimal, such as a syringe, topical application devices, or iv needletubing and bag, particularly in the case of the kits for producingspecific somatic animal models.

PCR-Based Methods for Identifying Novel AAV Sequences and Related Kits

Exemplary methods used for identifying novel AAV sequences are set forthbelow. Typically, the nucleic acids are used as primers in reversetranscription and/or polymerase chain reactions in the methods. Examplesof primers useful in a reverse transcription reaction include OligodT,random hexamers, and the sequence specific primers disclosed herein(e.g., SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4). Otherprimers appropriate for a reverse transcription reaction are known inthe art. Nucleic acids which are useful as PCR primers may have asequence that has substantial homology with a nucleic acid sequence of aregion that is highly conserved between at least two AAV serotypes. Insome cases, the region is highly conserved between two, three, four,five, six, seven, eight, nine, ten, eleven, or twelve or more AAVserotypes. Typically, the region that is highly conserved covers anend-to-end length of between 25 and 250 bp. In specific cases, theregion covers about 150 bp. However, in other cases the end-to-endlength of the highly conserved region is greater that 250 bp.Preferably, the region is highly conserved within this end-to-end lengthover at least about 9, and more preferably, at least 18 base pairs (bp).However, the region may be conserved over more than 18 bp, more than 25bp, more than 30 bp, or more than 50 bp.

In some embodiments the primers have a sequence as set forth in Table 1.

TABLE 1 AAV CAP GENE PRIMERS SEQ ID NUCLEIC ACID SEQUENCE SEQ ID NO: 1CapF-X(A/G/C/T/absent)GA(C/T)TG(C/T)(A/G/C)(A/T)(C/T/A)(A/T)(C/T)(G/T)GA(A/G)CAATAAATGA(A/G/C/T/absent) SEQ ID NO: 1 - CapF-XNGAYTGYVWHWYKGARCAATAAATGAN (single letter code) SEQ ID NO: 2 CapR-X(A/G/C/T/absent)GAAACGAAT(C/A/T)AA(C/A)CGGTTTATTGATTAA(A/ G/C/T/absent)SEQ ID NO: 2 - CapR-X NGAAACGAATHAAMCGGTTTATTGATTAAN (single lettercode) SEQ ID NO: 3 CapF GACTGTGTTTCTGAGCAATAAATGA SEQ ID NO: 4 CapRGAAACGAATTAACCGGTTTATTGATTAA SEQ ID NO: 5 CapF22-X(A/G/C/T/absent)(C/T)(C/A)(A/G)(T/A)(C/A)(A/G)(A/T)C(G/T)(G/T)(G/C)AGA(A/C)GCGG(A/G)(A/C)(G/C)(A/G/C/T/absent) SEQ ID NO: 5 CapF22-XNYMRWMRWCKKSAGAMGCGGRMSN (single letter code) SEQ ID NO: 6 CapF22CCATCGACGTCAGACGCGGAAG SEQ ID NO: 7 CapF64-X(A/G/C/T/absent)(G/C)(G/C)(C/A/G)GAC(A/G)(G/C)(G/C)T(A/C)(G/C)CA(A/G)(A/T)(A/T)CA(A/G)A(T/C)GT(A/G/C/T/absent) SEQ ID NO: 7 CapF64-XNSSVGACRSSTMSCARWWCARAYGTN (single letter code) SEQ ID NO: 8 CapF64GCCGACAGGTACCAAAACAAATGT SEQ ID NO: 8 CapF201-(A/G/C/T/absent)(C/A)(C/T)GG(C/A)(G/A)(T/C)GT(C/G)A(G/A)(A/T)AT(C/ XT)T(C/G)AA(C/T)C(A/G/C/T/absent) SEQ ID NO: 9 CapF201-NMYGGMRYGTSARWATYTSAAYCN (single letter X code) SEQ ID NO: 10 CapF201CCGGCGTGTCAGAATCTCAACC SEQ ID NO: 11 AV2cas-X(A/G/C/T/absent)AC(A/G)(C/G/T)(A/G)AGANCCAAAGTTCAACTGA(A/C)ACGA(A/G/C/T/absent) SEQ ID NO: 11 AV2cas-XNACRBRAGANCCAAAGTTCAACTGAMACGAN (single letter code) SEQ ID NO: 12AV2cas ACAGGAGACCAAAGTTCAACTGAAACGA

In some embodiments, the PCR methods comprise a first primer having thesequence as set forth in SEQ ID NO: 1 and a second primer having asequence as set forth in SEQ ID NO: 2. In some embodiments, the PCRmethods of the invention comprise a first primer having the sequence asset forth in SEQ ID NO: 3 and a second primer having a sequence as setforth in SEQ ID NO: 4.

The target sequence obtained in the PCR reaction may be all or a portionof the cDNA In some cases the cDNA is about 50, about 100, about 250,about 500, about 1000, about 2000, about 4000 base pairs in length. Incertain cases, the cDNA is approximately 2300 base pairs, approximately2600 base pairs, or approximately 4700 base pairs in length. However,the invention is not so limited and the actual cDNA length will dependon a variety of factors such as AAV serotype, RT reaction primers, RTreaction condition. In most cases, the cDNA has a length that issufficient to obtain unique sequence information that can be used toidentify the AAV serotype from which the amplified sequences originate.

The target sequence obtained in the PCR reaction may be all or a portionof one or more AAV rep or cap genes, such as VP1, VP2 and VP3.Alternatively, the target sequence obtained in the PCR reaction may beall or a portion of one or more AAV hypervariable regions. In the caseswhere a portion of a gene (e.g., VP1, VP2, or VP3) is obtained it isunderstood that the portion will be of a sufficient size and from anappropriate position within the gene (e.g., coding region, variableregion) to provide unique sequence information that can be used toidentify the AAV serotype from which the amplified sequences originate.

The PCR primers are generated using techniques known to those of skillin the art. Each of the PCR primer sets is composed of a forward primer(i.e., 5′ primer) and a reverse primer (i.e., 3′ primer). See, e.g.,Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (ThirdEdition). The term “primer” refers to an oligonucleotide which providesas a point of initiation of synthesis when placed under conditions (PCRreaction) in which synthesis of a primer extension product which iscomplementary to a nucleic acid strand is induced. The primer ispreferably single stranded. However, if a double stranded primer isutilized, it is treated to separate its strands before being used toprepare extension products. The primers may be about 15 to 30 or morenucleotides, and preferably at least 18 nucleotides. However, forcertain applications shorter nucleotides, e.g., 7 to 15 nucleotides areutilized. In certain embodiments, the primers are about 25 nucleotideslong (e.g., SEQ ID NO 3 or 4)

The primers are selected to be sufficiently complementary to thedifferent strands of each specific sequence to be amplified such thatthey hybridize with their respective strands. Typically, hybridizationoccurs under standard PCR conditions known in the art. Thus, primershaving melting temperatures between 50 and 65° C. are normally suitable.However, the invention is not so limited. In addition, the primersequence need not reflect the exact sequence of the region beingamplified. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer (e.g., for cloning purposes), withthe remainder of the primer sequence being substantially (e.g.,completely) complementary to the strand. In some cases, a primer mayinclude a sequence (e.g., 5′ sequence) that is not substantiallycomplementary to the target sequence but that facilitates subsequentmanipulation of the amplicon (e.g., cDNA). For example, in some cases, aprimer may have additional sequence at its 5′ end having a uniquerestriction site that facilitates subsequent digestion by an appropriaterestriction enzyme. Methods such as this can be employed to accomplish,for example, a cloning step. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to be amplified to hybridize therewith and form a template forsynthesis of the extension product of the other primer. Techniques suchas these and others disclosed herein are well known in the art and aresuitable for use in the methods of the instant invention.

The PCR primers for amplifying the target sequence according to theinvention are based upon the highly conserved sequences of two or morealigned sequences (e.g., two or more AAV serotypes). The primers canaccommodate less than exact identity among the two or more aligned AAVserotypes at the 5′ end or in the middle. However, the sequences at the3′ end of the primers correspond to a region of two or more aligned AAVserotypes in which there is exact identity over at least five,preferably, over at least nine base pairs, and more preferably, over atleast 18 base pairs at the 3′ end of the primers. Thus, the 3′ end ofthe primers is composed of sequences with 100% identity to the alignedsequences over at least five nucleotides. However, one can optionallyutilize one, two, or more degenerate nucleotides at the 3′ end of theprimer.

Both rep and cap gene transcripts are detected with variable abundancesby RNA detection methods (e.g., RT-PCR). The expression of cap genetranscripts and ability to generate cDNA of cap RNA through reversetranscription (RT) using the methods disclosed herein, significantlyincrease abundance of templates for PCR-based rescue of novel capsequences from tissues. The methods are useful for isolating novel fulllength functional cap cDNA sequences. The methods involve the design andselection of oligonucleotide primers for both RT and PCR reactions. Asdiscussed herein, AAV cap gene transcription is directed by AAV p40promoter which is located in the coding sequence of rep genes. Thus, insome cases, the region between the beginning of p40 RNA transcript andthe start codon of capsid VP1 cDNA is a target for the 5′ primers toretrieve the intact 5′ end of cap cDNA. In order to recover the intact3′ end of the cap transcript, the 3′ primer is typically selected in theregion of the polyadenylation signal. However, the invention is not solimited and other similar strategies can be employed to isolate novelcDNA sequences of this and other AAV genes.

In some cases, multiple primer sets are used to isolate novel cDNAsequences of an AAV gene in fragments. Fragments so obtained can, forexample, be cloned together to form a single cDNA comprising a completegene sequence. For example, a first primer set having a 5′ primercomplementary to an untranslated region of an AAV gene and a 3′ primer(anchor primer) complementary to a sequence within the AAV transcript(e.g., in an intronic or exonic sequence) can be used to obtain a firstfragment (e.g., a 5′ fragment of a gene sequence). A second primer set,having a 5′ primer (e.g., anchor primer) complementary to a sequenceupstream of the second 3′ primer of the first primer set and a 3′ primercomplementary to a position near the polyadenylation signal can be usedto obtain a second fragment (e.g., a 3′ fragment of a gene sequence).The two fragments can have any number of uses thereafter, for examplethey can be analyzed separately (e.g., sequenced) or cloned together toobtain a complete gene sequence. In some cases, three, four, five, sixor more primer sets can be used to obtain three, four, five, six or moreof AAV gene fragments. Moreover, these examples are not meant to belimiting and any number of primer sets can be employed to obtain anynumber of fragments provided that the fragments are useful foridentifying and obtaining unique AAV sequences (e.g., Capsid genesequences).

In some cases, the methods involve transfecting cells with totalcellular DNAs isolated from the tissues that potentially harbor proviralAAV genomes at very low abundance and supplementing with helper virusfunction (e.g., adenovirus) to trigger and/or boost AAV rep and cap genetranscription in the transfected cell. In some cases, RNA from thetransfected cells provides a template for RT-PCR amplification of cDNAand the detection of novel AAVs. In cases where cells are transfectedwith total cellular DNAs isolated from the tissues that potentiallyharbor proviral AAV genomes, it is often desirable to supplement thecells with factors that promote AAV gene transcription. For example, thecells may also be infected with a helper virus, such as an Adenovirus ora Herpes Virus. In a specific embodiment, the helper functions areprovided by an adenovirus. The adenovirus may be a wild-type adenovirus,and may be of human or non-human origin, preferably non-human primate(NHP) origin. Similarly adenoviruses known to infect non-human animals(e.g., chimpanzees, mouse) may also be employed in the methods of theinvention (See, e.g., U.S. Pat. No. 6,083,716). In addition to wild-typeadenoviruses, recombinant viruses or non-viral vectors (e.g., plasmids,episomes, etc.) carrying the necessary helper functions may be utilized.Such recombinant viruses are known in the art and may be preparedaccording to published techniques. See, e.g., U.S. Pat. Nos. 5,871,982and 6,251,677, which describe a hybrid Ad/AAV virus. A variety ofadenovirus strains are available from the American Type CultureCollection, Manassas, Va., or available by request from a variety ofcommercial and institutional sources. Further, the sequences of manysuch strains are available from a variety of databases including, e.g.,PubMed and GenBank.

Cells may also be transfected with a vector (e.g., helper vector) whichprovides helper functions to the AAV. The vector providing helperfunctions may provide adenovirus functions, including, e.g., E1a, E1b,E2a, E4ORF6. The sequences of adenovirus gene providing these functionsmay be obtained from any known adenovirus serotype, such as serotypes 2,3, 4, 7, 12 and 40, and further including any of the presentlyidentified human types known in the art. Thus, in some embodiments, themethods involve transfecting the cell with a vector expressing one ormore genes necessary for AAV replication, AAV gene transcription, and/orAAV packaging.

In some cases, a novel isolated capsid gene can be used to construct andpackage recombinant AAV vectors, using methods well known in the art, todetermine functional characteristics associated with the novel capsidprotein encoded by the gene. For example, novel isolated capsid genescan be used to construct and package recombinant AAV (rAAV) vectorscomprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase,etc.). The rAAV vector can then be delivered to an animal (e.g., mouse)and the tissue targeting properties of the novel isolated capsid genecan be determined by examining the expression of the reporter gene invarious tissues (e.g., heart, liver, kidneys) of the animal. Othermethods for characterizing the novel isolated capsid genes are disclosedherein and still others are well known in the art.

Kits are useful in some instances for practicing these methods in orderto rescue contaminates by helper AAV infections and/or detection oflatent virus that is transcriptionally active. Examples of such methodsare shown in the Examples and in particular Example 4.

The containers of the kit may house, for instance, any one or more ofthe following: at least one RNA detection component, at least one primerthat has substantial homology with a nucleic acid sequence that is about90% conserved between at least two AAV serotypes, at least one primerthat is substantially complementary to a nucleic acid sequencecorresponding to a 5′ or 3′ untranslated region of an AAV transcriptsuch as a transcript encoding a rep and/or cap gene, a set of PCRprimers specific for a signature region of the AAV nucleic acidsequence, a set of PCR primers specific for the full-length AAV capsidtranscript (i.e., the p40 intiated transcript), two or more additionalsets of primers, as described herein, and/or PCR probes, a primer havinga sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 4.

The kits may also include reagents for Reverse transcription componentsthat may include the following components: (a) at least one primer; (b)a Reverse Transcriptase (e.g., a Superscript); (c) nucleotides (e.g.,dNTPs); and (d) RT buffer. In some embodiments, the at least one primeris complementary to a portion of an AAV cDNA sequence. In someembodiments, the at least one primer is an OligodT primer. In someembodiments, the kits further comprise reagents for PCR components thatmay include the following components: (a) at least one primer; (b) athermostable polymerase (e.g., a Taq polymerase); (c) nucleotides (e.g.,dNTPs); and (d) PCR buffer. In some embodiments, the at least one primeris complementary to a portion of an AAV cDNA sequence. In someembodiments, the kits comprise a DNA isolation kit (e.g., Oragene,OG-100) and/or an RNA isolation kit (e.g., oligodT-cellulose columns).

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The instructions included within the kit may involve methods fordetecting a latent AAV in a cell. In addition, kits of the invention mayinclude, instructions, a negative and/or positive control, containers,diluents and buffers for the sample, sample preparation tubes and aprinted or electronic table of reference AAV sequence for sequencecomparisons.

EXAMPLES Example 1: Discovering New AAVs by Determining the Sequence ofAAV Transcripts Expressed from the Proviral Genome in Cells

The presence of Endogenous AAV transcripts in non-AAV transfer vectortreated Macaque tissues was examined by RT-PCR. PCR primers directedagainst Rep and Cap sequences were used for RT-PCR. DNase treated RNAsamples were used to control for amplification of contaminating genomicDNA having rep and cap sequences. No Reverse Transcription and No DNAsecontrols were included. Surprisingly, endogenous AAV transcripts aredetected in tissues of multiple non-AAV transfer vector treatedMacaques. The results indicate that endogenous proviral genomes of AAVare transcriptionally active.

A strategy was developed for designing primers useful for RT-PCRdetection of AAV RNA and identification novel AAV gene sequences such asthe capsid gene. Primers were selected in a highly conserved regionbetween multiple AAV genomes corresponding to the 5′ untranslated regionof a gene. A primer was designed in a highly conserved region betweenmultiple AAV genomes corresponding to the 5′ untranslated region of theVP1 gene and a portion of the VP1 open reading frame. The primer,identified as primer.CapF (i.e., SEQ ID NO 1 or 3), is a forward primerfor an AAV Cap gene.

Primers were selected in a highly conserved region between multiple AAVgenomes corresponding to the 3′ untranslated region of a gene. A primerwas designed in a highly conserved region between multiple AAV genomescorresponding to the 3′ untranslated region of the VP1, 2, and 3 geneand a portion of the VP1, 2, and 3 open reading frame. The primer,identified as primer.CapR (i.e., SEQ ID NO 2 or 4), is a reverse primerfor an AAV Cap gene. The primer, identified as AV2cas, is a reverseprimer for an AAV Cap gene.

Example 2: Isolation of Transcriptionally Active Novel AAV CapsidSequences from Chimpanzee Tissues for Vector Development

In an attempt to search for novel AAVs with propensities to infectprimates, a variety of chimpanzee tissues were analyzed for the presenceof AAV proviral genomes and cap RNAs first by qPCR and qRT-PCR using aset of primer and probe to target short stretches of the conserved capsequence. The data indicated that all the tissues harbored AAV invariable abundances, with the highest copy numbers in liver. Also, capgene was indeed transcriptionally active and cap RNAs were detected inall the samples, but levels of cap RNA were more consistent amongdifferent tissues with copy numbers, generally speaking, higher than DNAsequences. Subsequently, PCR and RT-PCR cloning were undertaken torescue full length capsid sequences from both chimpanzee DNAs and RNAs.A total of 48 cDNA and 28 DNA clones of VP1 were analyzed by sequencing.The phylogenetic analysis segregated those clones into three majorgroups closely related to AAV1, AAV6 and AAV9 which hold promises forgene delivery to lung, CNS and skeletal and cardiac muscle targets,respectively, in murine, canine and NHP models. Further analysis ofthose clones led to several key findings. First, in any tissues whereboth cDNA and DNA clones were generated, the capsid sequences of DNA andcDNA origins are phylogenetically different. While all the clones ofAAV1 relatives were derived from chimpanzee cellular DNAs, all ofAAV6-like and a majority of AAV9-like clones were the products ofRT-PCR. Secondly, full length cap RT-PCR did not recover products fromchimp spleen and heart, although the copy numbers of the transcripts inthose two tissues by qRT-PCR were similar to those of other tissues.Finally, only cap cDNA, not DNA, clones were isolated from brain andlung tissues. A subset of the novel AAV clones were selected on thebasis of their sequence distinctiveness from their AAV1, AAV6 and AAV9relatives for further evaluation of vector packaging, tissue tropism,gene transfer efficiency and stability, sero-prevalence, vector-relatedtoxicity and pathology, capsid and transgene T cell, etc. Dendrogramsdepicting the results of hierarchical cluster analyses of the new AAVsare provided in FIGS. 5-9. Table 2 provides a listing of the BLASTresults using the sequences of the isolated AAVs as query sequences. Theaccession numbers and AAV serotypes of the best match sequences areprovided. Fractions of identical amino acids and gap sites are alsoprovided. Table 3A provides the group number of each variant based onnucleic acid alignment, the name of each variant, the tissue from whicheach variant was isolated, an indication of whether a variant wasisolated from genomic DNA (gDNA) or using RT-PCR (cDNA); the length ofthe DNA and translated protein sequences of each variant; and thecorresponding SEQ ID NOs. DNA sequences of Csp-3, Csp-7, Ckd-B6, Ckb-B8,CLv-D6, CLv-D7, Clg-f1, Clg-f8, Csp-10, Csp-4, Ckd-4, CLv-12, Clv-3contained mutation(s) that led to impaired VP1 protein translation.Those sequences were repaired manually before they could be translatedinto a complete VP1 protein sequences. A listing of the SEQ ID NOs ofthe non-repaired DNA sequences is provided in Table 3B. Table 3C listsadditional AAV9 variants discovered in chimpanzee tissues. Table 3Dlists AAV serotypes of Table 3A and their corresponding SEQ ID NOs.Table 4A-C provides a comparison of certain corresponding amino acidsamong example AAV variants and their related AAV serotypes.

TABLE 2 BLAST RESULTS USING - NOVEL AAV QUERY SEQUENCES Best match foundwith blastp in GB query accession name identity gaps CKd-B6 AAB95450.1AAV6 729/736 0/736 CKd-B1 AAB95450.1 AAV6 732/736 0/736 CKd-B2AAB95450.1 AAV6 733/736 0/736 CBr-E6 AAS99264.1 AAV9 732/736 1/736CLv-D2 AAS99264.1 AAV9 731/736 0/736 CLv-D8 AAS99264.1 AAV9 732/7360/736 CLv-R1 AAS99264.1 AAV9 732/736 0/736 CLv-R7 AAS99264.1 AAV9728/736 0/736 CLg-F3 AAS99264.1 AAV9 731/736 0/736 CLg-F4 AAS99264.1AAV9 730/736 0/736 CLg-F7 AAS99264.1 AAV9 732/736 0/736 CSp-1 AAS99264.1AAV9 732/736 0/736 CSp-3 AAS99264.1 AAV9 730/736 0/736 CSp-7 AAS99264.1AAV9 732/736 0/736 CBr-E1 = AAS99264.1 AAV9 734/736 0/736 CLv-E1 CBr-E2AAS99264.1 AAV9 734/736 0/736 CBr-E3 AAS99264.1 AAV9 735/736 0/736CBr-E4 AAS99264.1 AAV9 735/736 0/736 CBr-E5 AAS99264.1 AAV9 733/7360/736 CBr-e5 AAS99264.1 AAV9 734/736 0/736 CBr-E7 AAS99264.1 AAV9734/736 1/736 CBr-E8 AAS99264.1 AAV9 734/736 0/736 CLv-D1 AAS99264.1AAV9 733/736 0/736 CLv-D3 AAS99264.1 AAV9 734/736 0/736 CLv-D4AAS99264.1 AAV9 734/736 0/736 CLv-D5 AAS99264.1 AAV9 734/736 0/736CLv-D6 AAS99264.1 AAV9 735/736 0/736 CLv-D7 AAS99264.1 AAV9 732/7360/736 CLv-R2 AAS99264.1 AAV9 734/736 0/736 CLv-R3 AAS99264.1 AAV9735/736 0/736 CLv-R4 AAS99264.1 AAV9 734/736 0/736 CLv-R5 AAS99264.1AAV9 733/736 0/736 CLv-R6 AAS99264.1 AAV9 736/736 0/736 CLv-R8AAS99264.1 AAV9 735/736 0/736 CLv-R9 AAS99264.1 AAV9 730/736 0/736CLg-F1 AAS99264.1 AAV9 730/736 0/736 CLG-F2 AAS99264.1 AAV9 732/7360/736 CLg-F5 = AAS99264.1 AAV9 734/736 0/736 CLg-F6 = CLg-F8 CSp-10AAS99264.1 AAV9 735/736 0/736 CSp-11 AAS99264.1 AAV9 733/736 0/736 CSp-2AAS99264.1 AAV9 733/736 0/736 CSp-4 AAS99264.1 AAV9 733/736 0/736 CSp-6AAS99264.1 AAV9 733/736 0/736 CSp-8 AAS99264.1 AAV9 735/736 0/736 CSp-9AAS99264.1 AAV9 734/736 0/736 CKd-H2 ABA71701.1 AAV.VR-355 727/732 0/732(AAV6 like) CKd-B3 ACB55301.1 AAV6.1 729/736 0/736 CKd-B8 ACB55301.1AAV6.1 732/736 0/736 CKd-B4 ACB55301.1 AAV6.1 731/736 0/736 CKd-B5 =ACB55301.1 AAV6.1 734/736 0/736 CKd-H6 CKd-B7 ACB55301.1 AAV6.1 730/7360/736 CKd-H1 ACB55301.1 AAV6.1 732/736 0/736 CKd-H3 ACB55301.1 AAV6.1733/736 0/736 CKd-H4 ACB55301.1 AAV6.1 732/736 0/736 CKd-H5 ACB55301.1AAV6.1 731/736 0/736 CKd-3 ACB55310.1 AAV.hu.48R3 735/736 0/736 CKd-1NP_049542.1 AAV1 733/736 0/736 CKd-7 NP_049542.1 AAV1 731/736 0/736CLv-4 NP_049542.1 AAV1 733/736 0/736 CHt-2 NP_049542.1 AAV1 734/7360/736 CHt-3 NP_049542.1 AAV1 735/736 0/736 Ckd-10 NP_049542.1 AAV1734/736 0/736 CKd-2 NP_049542.1 AAV1 734/736 0/736 CKd-4 NP_049542.1AAV1 733/736 0/736 CKd-6 NP_049542.1 AAV1 733/736 0/736 CKd-8NP_049542.1 AAV1 734/736 0/736 CLv-1 NP_049542.1 AAV1 734/736 0/736CLv-12 NP_049542.1 AAV1 733/736 0/736 CLv-13 NP_049542.1 AAV1 734/7360/736 CLv-2 NP_049542.1 AAV1 734/736 0/736 CLv-3 NP_049542.1 AAV1730/736 0/736 CLv-6 NP_049542.1 AAV1 734/736 0/736 CLv-8 NP_049542.1AAV1 735/736 0/736 CHt-1 YP_680426.1 AAV2 734/735 0/735 Total proteins78 Non-redundant 74 CBr-E1 = CLv-E1 CLg-F5 = CLg-F6 = CLg-F8 CKd-B5 =CKd-H6 Table 3A outlines the properties of isolated AAVs and providesSEQ ID NOS for each AAV Table 3B lists examples of unedited DNAsequences Table 3C provides the protein sequences of additional isolatedAAV9 variants.

TABLE 3A SEQUENCES OF NOVEL AAVS Protein Group length (based on gDNA DNADNA (aa, amino PROTEIN DNA or length SEQ ID acid) - SEQ ID alignment)Name Tissue cDNA (bp) NO: Predicted NO: 1 CBr-E1 Brain cDNA 2208 13 73687 1 CBr-E2 Brain cDNA 2208 14 736 88 1 CBr-E3 Brain cDNA 2208 15 736 891 CBr-E4 Brain cDNA 2208 16 736 90 1 CBr-E5 Brain cDNA 2208 17 736 91 1CBr-e5 Brain cDNA 2208 18 736 92 1 CBr-E6 Brain cDNA 2205 19 735 93 1CBr-E7 Brain cDNA 2205 20 735 94 1 CBr-E8 Brain cDNA 2208 21 736 95 1CLv-D1 Liver cDNA 2208 22 736 96 1 CLv-D2 Liver cDNA 2208 23 736 97 1CLv-D3 Liver cDNA 2208 24 736 98 1 CLv-D4 Liver cDNA 2208 25 736 99 1CLv-D5 Liver cDNA 2208 26 736 100 1 CLv-D6 Liver cDNA 2209 27 736 101 1CLv-D7 Liver cDNA 2208 28 736 102 1 CLv-D8 Liver cDNA 2208 29 736 103 1CLv-E1 Liver cDNA 2208 13 736 87 1 CLv-R1 Liver cDNA 2208 30 736 104 1CLv-R2 Liver cDNA 2208 31 736 105 1 CLv-R3 Liver cDNA 2208 32 736 106 1CLv-R4 Liver cDNA 2208 33 736 107 1 CLv-R5 Liver cDNA 2208 34 736 108 1CLv-R6 Liver cDNA 2208 35 736 109 1 CLv-R7 Liver cDNA 2208 36 736 110 1CLv-R8 Liver cDNA 2208 37 736 111 1 CLv-R9 Liver cDNA 2208 38 736 112 1CLg-F1 Lung cDNA 2207 39 735 113 1 CLg-F2 Lung cDNA 2208 40 736 114 1CLg-F3 Lung cDNA 2208 41 736 115 1 CLg-F4 Lung cDNA 2208 42 736 116 1CLg-F5 Lung cDNA 2208 43 736 117 1 CLg-F6 Lung cDNA 2208 43 736 117 1CLg-F7 Lung cDNA 2208 44 736 118 1 CLg-F8 Lung cDNA 2208 43 736 117 1CSp-1 Spleen gDNA 2208 45 736 119 1 CSp-10 Spleen gDNA 2207 46 735 120 1CSp-11 Spleen gDNA 2208 47 736 121 1 CSp-2 Spleen gDNA 2208 48 736 122 1CSp-3 Spleen gDNA 2207 49 735 123 1 CSp-4 Spleen gDNA 2208 50 736 124 1CSp-6 Spleen gDNA 2208 51 736 125 1 CSp-7 Spleen gDNA 2208 52 736 126 1CSp-8 Spleen gDNA 2208 53 736 127 1 CSp-9 Spleen gDNA 2208 54 736 128 2CHt-2 Heart gDNA 2208 55 736 129 2 CHt-3 Heart gDNA 2208 56 736 130 2CKd-1 Kidney gDNA 2208 57 736 131 2 Ckd-10 Kidney gDNA 2208 58 736 132 2CKd-2 Kidney gDNA 2208 59 736 133 2 CKd-3 Kidney gDNA 2208 60 736 134 2CKd-4 Kidney gDNA 2208 61 736 135 2 CKd-6 Kidney gDNA 2208 62 736 136 2CKd-7 Kidney gDNA 2208 63 736 137 2 CKd-8 Kidney gDNA 2208 64 736 138 2CLv-1 Liver gDNA 2208 65 736 139 2 CLv-12 Liver gDNA 2208 66 736 140 2CLv-13 Liver gDNA 2208 67 736 141 2 CLv-2 Liver gDNA 2208 68 736 142 2CLv-3 Liver gDNA 2207 69 735 143 2 CLv-4 Liver gDNA 2208 70 736 144 2CLv-6 Liver gDNA 2208 71 736 145 2 CLv-8 Liver gDNA 2208 72 736 146 3CKd-B1 Kidney cDNA 2208 73 736 147 3 CKd-B2 Kidney cDNA 2208 74 736 1483 CKd-B3 Kidney cDNA 2208 75 736 149 3 CKd-B4 Kidney cDNA 2208 76 736150 3 CKd-B5 Kidney cDNA 2208 77 736 151 3 CKd-B6 Kidney cDNA 2208 78736 152 3 CKd-B7 Kidney cDNA 2208 79 736 153 3 CKd-B8 Kidney cDNA 221180 736 154 3 CKd-H1 Kidney cDNA 2208 81 736 155 3 CKd-H2 Kidney cDNA2196 82 732 156 3 CKd-H3 Kidney cDNA 2208 83 736 157 3 CKd-H4 KidneycDNA 2208 84 736 158 3 CKd-H5 Kidney cDNA 2208 85 736 159 3 CKd-H6Kidney cDNA 2208 77 736 151 4 CHt-1 Heart gDNA 2205 86 735 160

TABLE 3B EXAMPLE UNEDITED SEQUENCES OF ISOLATED AAVS Group (based on DNAgDNA or DNA alignment) Name Tissue cDNA SEQ ID NO: 1 CLv-D6 Liver cDNA161 1 CLg-F1 Lung cDNA 162 1 CLg-F8 Lung cDNA 163 1 CSp-10 Spleen gDNA164 1 CSp-3 Spleen gDNA 165 1 CSp-4 Spleen gDNA 166 2 CKd-4 Kidney gDNA167 2 CLv-12 Liver gDNA 168 2 CLv-3 Liver gDNA 169 3 CKd-B8 Kidney cDNA170

TABLE 3C Additional AAV9 Variants Name (a.a. length) SEQ ID NO: CLv1-1(736) 171 CLv1-2 (736) 172 CLv1-3 (736) 173 CLv1-4 (736) 174 CLv1-7(736) 175 CLv1-8 (736) 176 CLv1-9 (736) 177 CLv1-10 (736) 178

TABLE 3D AAV CAPSID SEQUENCES GenBank SEQ ID NO's of Name AccessionNumber SEQ ID NO VARIANTS AAV6 AAB95450.1 179 147, 148, 152 AAV9AAS99264.1 180 87-128 AAV.VR-355 ABA71701.1 181 156 (AAV6 like) AAV6.1ACB55301.1 182 149-151, 153-155, 157-159 AAV.hu.48R3 ACB55310.1 183 134AAV1 NP_049542.1 184 129-133 or 135-146 AAV2 YP_680426.1 185 160

TABLE 4A Amino Acid Differences of Example AAV9 Variants Amino Acid AAV9(SEQ Csp3 (SEQ CLv-D8 (SEQ Clg-F1 (SEQ Position ID NO 178) ID NO: 123)ID NO 103) ID NO: 113) 74 E E E V 93 Y Y Y C 203 M I M M 259 Q R Q Q 275F F F L 321 Q R Q Q 335 A T A A 373 M M M T 445 Y Y H Y 495 Q R Q Q 527H H Y H 533 R R S R 639 M V M M 647 I I T I 729 T T T P 736 L L L F

TABLE 4B Amino Acid Differences of an Example AAV1 Variant Amino AcidAAV1 CkD-7 Position (SEQ ID NO: 182) (SEQ ID NO: 137) 38 K E 599 M T 601A T 651 N T 717 N D

TABLE 4C Amino Acid Differences of Example AAV6.1 Variants Amino AcidAAV6.1 (SEQ Ckd-B7 (SEQ Ckd-B8 (SEQ Position ID NO: 180) ID NO: 153) IDNO: 154) 7 L F L 161 K R K 260 I I T 418 D E E 496 N N Y 584 L F F 589 TP T 722 T S T

Example 3: Isolation and Characterization of Novel Cap cDNAs from thePrimate Brain and Other Tissues that are Naturally Infected with AAV9Variants

Naturally occurring AAV9 variants that are capable of escaping from hostimmune defenses and are transcriptionally active, serve as goodsubstrates for retrieval of novel cap cDNA. Recent results revealed thepresence of both rep and cap gene transcripts in rhesus and cynomolgusmacaque tissues. Results disclosed herein corroborate these findings inchimpanzee tissues and provide a panel of novel AAV capsid cDNA clonesthat group with AAV9 Glade phylogenetically. Brain and other tissuesfrom chimpanzees were analyzed for cap RNA transcripts, and cap cDNAclones were isolated and analyzed by RT-PCR cloning for sequencecharacterization. The cap cDNA clones with 4 or more amino aciddifferences from AAV9 and within themselves were selected for furthercharacterization, based on their phylogenetic grouping and vectorproductivity. (See Tables 3A-C).

Transcriptional activity of rep/cap genes in the AAV DNA positive NHPtissues was evaluated by RT-PCR analysis. The presence of rep/captranscripts in some NHP tissues was confirmed. Next, cap cDNAs from NHPtissues were isolated for vector development purposes, aiming toidentify novel capsid sequences that are phylogenetically close to AAV9,similar or better in crossing the BBB via intravascular delivery but,importantly, more immunologically suitable for stable and efficient genetransfer. To this end, considering phylogenetic and physiologicalcloseness of chimpanzees to humans as well as recent work in which thechimp was found to be a better model in some instances to predictperformance of AAV vectors in human airway epithelial cells, 6 tissues(brain, heart, kidney, liver, lung and spleen) were evaluated from twochimps for AAV cap sequences. The data indicated that all the tissuesharbored AAV in variable abundances, with the highest copy numbers inthe liver. Also, cap gene was indeed transcriptionally active and capRNAs were detected in all the samples. The levels of cap RNAs wereconsistent among different tissues with the copy numbers higher than DNAsequences.

Subsequently, PCR and RT-PCR cloning was undertaken to rescue fulllength capsid sequences from both chimpanzee DNAs and RNAs respectively.A total of 48 cDNA and 28 DNA clones of VP1 were isolated by RT-PCR andPCR and fully sequenced. The phylogenetic analysis segregated thoseclones into three major groups that are closely related to AAV1, AAV6and AAV9, which hold promises for gene delivery to lung, skeletal andcardiac muscle, and CNS targets, respectively. Interestingly, 45 out ofthese 76 clones were AAV9-like, of which 35 clones were the products ofRT-PCR. A subset of the novel AAV9-like clones were selected for furtherevaluation on the basis of their sequence predicted structuraldistinctiveness from AAV9. The selected clones are comprised of 12 cDNAclones and 3 DNA clones with 4-6 amino acid differences from the capsequence of AAV9. Thus, feasibility of isolating cap cDNAs of novel AAV9variants from the transcriptionally active AAV genomes resided in NHPtissues was demonstrated.

Using a web-based protein structure homology modeling program, theSwiss-Model, AAV9 VP3 structure was predicted using the published AAV8crystal structure as the reference, where the regions with biggestdifferences between AAV8 and AAV9 were identified. (Arnold K, Bordoli L,Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environmentfor protein structure homology modelling. Bioinformatics 2006;22:195-201). Each of those 15 capsid sequences of AAV9 variants werecompared with that of AAV9 and identified a total of 54 amino acidchanges in which 37 changes are located in the predicted AAV9 VP3crystal structure. Interestingly, some of those changes are found in theT cell epitopes and proposed receptor binding domains identifiedpreviously, suggesting potential differences in the vector biologyincluding T cell immunological profiles (3, 17, 18).

Example 4: Characterization of Isolated AAVs

More than 50 full length capsid sequences that are closely related toAAV9 were isolated from chimpanzee tissues. A subset of these novel AAV9variants that were retrieved from either cellular DNA by PCR (Csp3 andCsp7) or RNA by RT-PCR (ClgF1, ClgF4, ClvD8, ClvR7 and ClvR9) wereselected for vector development and evaluation. Relationships betweencapsid structure and vector biology, and production yield wereinvestigated; a summary of this characterization is provided as Table 5.The vectors were evaluated in C57BL/6 mice for nLacZ gene transductionof liver, heart, pancreas, skeletal, muscle and lung (FIG. 12-13).Differences in capsid sequences between AAV9 and these seven novelvariants range from 4 to 6 amino acids. Three vectors were packagedefficiently whereas the other four were not, suggesting that severalamino acids are critical for AAV9 packaging. Those three vectors wereevaluated in C57BL/6 mice for nLacZ and α1-antitrypsin (A1AT) genetransduction after intravenous (i.v.), intramuscular (i.m.), andintranasal (i.n.) administration (FIG. 14).

A study comparing nLacZ transduction in liver, skeletal muscle and lungwas performed. The results of this study suggested that Csp3 outperformsAAV9. In the lung, both Csp3 and ClvD8 primarily target alveoli as doesAAV9 (FIG. 15.) However, data from quantitative comparisons at earlytime points using A1AT as a reporter gene demonstrated no significantdifferences in liver and muscle transduction between AAV9 and Csp3. Aslight increase by Csp3 compared with AAV9 was observed in A1ATtransduction in lung after intranasal delivery. Additionally, whileClgF1 vector lead to poor expression of both transgenes (nLacz and A1AT)in all target tissues, the ClvD8 effectively transduces muscle and lung.

Using newly established crystal structure of AAV9 VP3 as the model, thecapsid structure of Csp3, which differs from AAV9 in 6 amino acidresidues, with that of AAV9. Of these six amino acids, the methionine atamino acid position 203 is conserved, and although not present in thecrystal structure, should be located inside near the 5 fold pore and mayplay a role in AAV transduction. The glutamine at amino acid position259 is located at the subunit interface of monomers that form pentamers.The glutamine at amino acid position 321, which is near the base of5-fold pore, is in a highly conserved region of the capsid, as is thealanine at amino acid position 335, which are inside the 5-fold channel.The glutamine at amino acid position 495 and the methionine at aminoacid position 640 are located on the surface of 3-fold symmetry axis.

TABLE 5 Yield and Transduction Characterization of AAV Variants Aminoacid Productivity A1AT expression Name of difference (×10e13 GC/ml)nLacZ transduction (% AAV9) (4 w) (ng/ml) variant from AAV9 nLacZ EGFPA1AT Liver Heart Muscle Lung i.v. i.m. i.n. AAV9 0 2 2 0.8 ++++ +++++++++ +++ 100    100    100    (1.28E+08) (8.61E+06) (2.12E+03) Csp-3 6 10.7 0.8 +++++ +++++ +++++ ++++ 16.2  26.3  61.8 Csp-7 4 0.06 ND ND + +/−ND ND ND ND ND Clg-F1 6 0.6 1 0.5 + +/− + +   0.0002  0.008 14.5 Clg-F46 0.028 ND ND + +/− ND ND ND ND ND Clv-D8 4 0.27 0.7 0.5 + +/− ++ +++2.8 0.2 26.6 Clv-R7 8 NP ND ND ND ND ND ND ND ND ND Clv-R9 6 NP ND ND NDND ND ND ND ND ND Clv-1.9 4 ND ND 0.4 ND ND ND ND 0.1 0.2 34.4 Clv-1.104 ND ND  0.45 ND ND ND ND  0.03 0.4 31.4

Example 5: Vector Creation and Screening for Vector ProductivityIsolated AAV9 Variants

The Blood-Brain-Barrier (BBB) is an important cellular and metabolicstructure in the central nervous system (CNS). For the treatment of manyCNS disorders, this important structure becomes a barricade thatprevents the therapeutics from entering the CNS. Recombinantadeno-associated virus (rAAV) mediated gene transfer is an attractivestrategy for treating CNS diseases. However, development of the rAAVbased effective CNS gene therapeutics has been hindered by thedeficiency of the earlier generations of vectors in crossing the BBB andglobally delivering the genes to the CNS.

AAV vectors are created from novel AAV9 variants that have already beenisolated and tested for packaging efficiency. Cap cDNAs are isolated ofAAV9 variants from other primate tissues including macaques and humansthrough optimization of primer design and PCR conditions. The new capcDNA clones are sequenced and more clones are selected that fall intoAAV9 clades phylogenetically but have some structurally uniqueness fortesting vector production. Considering high doses of vector are oftenrequired for intravascular delivered gene therapeutics to target the CNSvector production is often an important aspect of clinical vectordevelopment. Thus, vector titer and yield are among the criteria usedfor candidate selection. An objective is to identify novel AAV9 variantsfor large scale vector production and vector biology evaluation.

Another consideration in the vector creation and biological evaluationin vivo is the genome format of the reporter gene vector. Capsidstructure plays a role in both cellular uptake and intracellulartrafficking that lead to transduction. But the conversion of thetranscriptionally inactive single stranded genomes to thedouble-stranded form depicts a remarkable post entry block (19). As acomponent of the analysis of vector biology on the capsid, the EGFPexpressing self-complementary vector genome is used to produce vectors,which bypasses the genome conversion process and initiate transductionimmediately after entering the nuclei of the target cells.

Since the capsid is a determinant of the AAV vector biology, atrans-encapsulation method is employed to package rAAV2 genomes with thecapsids from naturally occurring AAV9 variants that are isolated.Briefly, chimeric packaging constructs are created by ligating the AAV2Rep gene with Cap genes of novel AAVs at an Xho I site through a partialdigestion. The first Xho I site in the AAV2 genome is located just atthe very beginning of the VP1 gene where high homology among all knownAAV sequences is observed. This Xho I site is present in AAV sequenceswhich have been analyzed and has been used for constructing hybridpackaging plasmids of more than 50 novel AAVs, as previously reported(8).

As the first step of lead vector selection, hybrid packaging plasmidsare assessed for their vector packaging efficiency in small scale testproduction. AAV cis plasmids carrying the self-complementary AAVEGFP(scAAVEGFP) genome are transfected into 293 cells in 6 well platestogether with the chimeric packaging plasmids containing novel AAVcapsid gene and adenovirus helper plasmid. AAV9 packaging plasmid isused as a control. Seventy-two hours later, crude cell lysate isharvested and subjected to 3 cycles of freezing and thawing. Seriallydiluted crude cell lysates are put on 293 cells in a 24 well plate inthe presence of adenovirus helper. Numbers of EGFP positive cellsresulting from new chimeric packaging plasmids are estimated andcompared with that of AAV9.

A further criterion for candidate vector selection is that packagingplasmids should have scAAVEGFP productivity (as measured by 293 celltransduction) typically at a level no less than 10% of that produced byusing AAV9 packaging plasmid for the large scale AAV vector production.The vectors are produced by the same protocol as used for the testproduction and are purified by standard CsCl₂ sedimentation method.Genome titers of purified vector preps are determined by real time PCR.The purity of the vector preps is examined by sliver stained SDS-PAGEand negative stained Electron Microscopy.

Isolation of More AAV9 Variants from Other Primate Tissue Sources forVector Creation

As indicated herein, available clones of cap cDNA from novel AAV9variants are available from chimpanzee tissues. This collection of capcDNAs of AAV9 relatives is expanded by isolating cap cDNAs in otherprimate tissues, particularly brain tissues. Without wishing to be boundby theory, brain tissue derived endogenous AAVs may have crossed theBlood-Brain Barrier (BBB) and established latency in the brain duringnatural infections. Endogenous AAVs that are transcriptionally active inhuman tissues may have the immunological properties more suitable forthe human applications. In previously reported studies on novel AAVdiscovery, approximately 474 macaque tissues and 259 human tissues werecollected with IRB approval (7). The same collection of frozen tissuesis used for RNA extraction and recovery of cap cDNAs.

In this regard, primer design and RT-PCR conditions are optimized toenrich recovery of the cap cDNAs that fall into the AAV9 Glade. In theinitial isolation of novel cap cDNAs from chimpanzee tissues, universalprimers were designed that could anneal to the 5′ and 3′ conservativeregions of the cap genes of all available AAV sequences, aiming toamplify as many types of AAV sequences as possible. Depending onspecific AAV sequences, sequence homology between the universal primersand a particular AAV sequence may vary. This may be compensated by usingless stringent PCR conditions such as annealing temperatures,annealing/extension time and numbers of amplification cycles. Thisdesign strategy is effective to retrieve cap cDNA of all different AAVsbut may not be sufficient to target and rescue the sequences that arecloser to AAV9, in all cases. Using routine methods, primers aredesigned that have better matches with AAV9. The new primers are testedfor the detection sensitivity and amplification efficiency underdifferent RT-PCR conditions, using different RT-PCR reagents and theserially diluted AAV9 clone as the template. Different parameters thatare evaluated include one-step RT-PCR versus two-step RT-PCR, oligo-dTversus random priming for cDNA creation, different reversetranscriptases, Taq polymerases and associated reagents, etc.

A next step in isolating the full length cap cDNA clones is thepreparation of high quality of cellular RNAs with minimum cellular DNAcontamination. This is accomplished by TRIzol-based tissue RNAextraction followed by the treatment with RNase-free DNase andpurification using a column from the Qiagen RNeasy kit. This methodremoved cellular DNA contamination effectively as demonstrated by thelack of cap PCR band in the corresponding RT (−) reaction for each RT(+) PCR reaction. Leftover frozen primate tissues which were positivefor endogenous AAV by cap PCR screening previously (19% of 474 NHPtissues and 18% of 259 human tissues) (7) are subjected to cellular RNAextraction and RT-PCR screening using the reagents and conditionsoptimized according to the methods disclosed herein. The RT-PCR productsare cloned (e.g., TOPO-cloned) for sequencing characterization.

The sequences of new cap cDNA clones are compared with all available AAVsequences in the GenBank. Clones that fall into the AAV9 Gladephylogenetically are chosen for redundancy assessment. The AAV9 variantsfrom the same tissue and/or subject with less than 4 cap residuedifferences are considered as redundant (8). Newly isolatednon-redundant clones of AAV9 variants are converted into chimericpackaging plasmids and first screened for AAV vector production asdescribed above. A goal is to select variants that meet the acceptableproductivity standards for the vector biology evaluation in vitro and invivo.

Variations may occur in the yields of transcapsulated reporter genevectors with different capsid variants in terms of total vector genomecopies (GCs) as well as transduction titers when measured in vitro. AAVvector development has suggested a lack of correlation between in vitroand in vivo transduction. AAV9 vectors are produced routinely ofdifferent transgenes at yields in the ranges of 0.5-1×10⁵ vector genomesper cell. Novel vectors with the yields less than 1×10⁴ GCs/cell are nottypically pursued. It is possible that the deficiency in vectorpackaging and transduction of some vectors could be caused by theincompatibilities between vector ITRs, rep, cap sequences and the originof adenovirus helper genes. An alternative strategy is to rescue theinfectious molecular clones of the endogenous AAV genomes from theoriginal tissues and develop vectors in which ITRs, rep and capsequences are derived from the same virus isolate (8). For thechimpanzee tissue derived AAV9 variants, helper genes from chimpanzeeadenoviruses may be required for efficient vector packaging. In thiscase, molecular clones of chimpanzee adenoviruses may be modified fortheir application in the AAV vector production (20).

Selection of Lead Candidate Vectors (LCVs) and Clinical CandidateVectors (CCVs) in Murine and Chimp Models

A family of novel AAVs exists in NHPs and humans with structural andfunctional similarity to AAV9 Glade and unique immunological propertiesthat are candidates for global delivery of gene therapeutics to CNSintravascularly.

In addition to its superb transvascular gene transfer efficiency inheart and muscle, AAV9-based vector has demonstrated a unique capabilityto cross the BBB and lead to neuronal and neuroglial gene transfer inneonatal mice and astrocyte gene transfer in adult mice (9). CCVs aresought to be identified from candidates clones of AAV9 variants thathave improved the immunological properties with respect to serologicalprevalence, capsid and transgene directed T cell immunity. Using AAV9vector as the bench marker, LCVs are selected from starting clones ofthe novel AAV9 variants in vitro and in C57BL/6 mice for theircapability to escape from serological barriers in pooled human IGIVs andto cross the BBB to achieve global CNS gene transfer. The LCVs with AAV9are further compared in two strains of mice (C57BL/6 and BALB/c) for thecell type tropism, transduction efficiency and stability, dose response,vectortoxicity, and non-CNS tissue targeting. Gene transfer to asurrogate tissue of the chimpanzees, vector toxicity andtransgene/capsid immunities in chimps is also tested.

Experimental Design

To identify CCVs from candidate vectors based on their biologicalmerits, an algorithm is designed to enable CCVs selection in a datadriven and quantitative manner. In this design, a total of 15 differentparameters capturing all relevant aspects of the vector biology areevaluated at different stages of screening using a quantitative scoringsystem as summarized in Table 6. A score of 0, 1, 2, or 3 is used toassess each parameter. The specific scores based on the grading criteriaare also presented in Table 6. The lower number represents the morefavorable vector biology. In some cases, these 15 parameters aredistilled down to the following 5 criteria: Preexisting immunity (invitro and in vivo neutralization antibody (NAB)), gene transfer(efficiency, cell type tropism and stability), non-CNS tissue targeting(liver, heart and pancreas gene expression and vector genomebiodistribution), toxicity [Liver function tests (LFTs), brain and liverhistopathologies] and immunology (capsid and transgene T cells andvector capsid antibody). The rationale for combining some of theparameters into specific criteria such as LFTs and histopathology into“toxicity” are: 1) they represent the same biology measured differentways, and 2) provides equal weight in the final analysis for the basic 5criteria.

TABLE 6 Algorithms for Selection of LCVs and CCVs Rating ObjectiveParameter Measurement 0 1 2 3 I. Selection of 3 LCVs from 15 candidatevectors 1). Pre-existing Immunity/hIVIG In vitro/NAB titer AAVx/AAV9 <1x =1x  2-4x >10x   Passive transfer hIGIV dose (mg)-inhibition ≥40  ≥12   ≥4 ≥1.2 2). CNS gene transfer in C57BL/6 mice by the clonespassing serological tests - 1 litter of neonates and 6 male adults pervector Spinal cord - total EGFP+ AAVx/AAV9 >1 0.5-1   0.25-0.5 <0.1Brain - total EGFP+ AAVx/AAV9 >1 0.5-1   0.25-0.5 <0.1 II. Selection of2 CCVs from 3 LCVs and AAV9 in C57BL/6 and BALB/c mice - 3 doses, 2 timepoints, 1 litter of neonates and 6 male adults/vector/dose/time point1). Gene transfer Spinal cord % EGFP(+)/ChAT(+) AAVx/AAV9 >1 0.5-1  0.25-0.5 <0.1 % EGFP(+)/GAFP(+) AAVx/AAV9 >1 0.5-1   0.25-0.5 <0.1 Brain% EGFP(+)/NeuN(+) AAVx/AAV9 >1 0.5-1   0.25-0.5 <0.1 % EGFP(+)/GAFP(+)AAVx/AAV9 >1 0.5-1   0.25-0.5 <0.1 EGFP-Stability in CNS Spinal Cord Day90/Day 14   >0.75 0.5-0.75 0.25-0.5  <0.25 Brain Day 90/Day 14   >0.750.5-0.75 0.25-0.5  <0.25 2). Non-CNS tissue targeting Liver - EGFP+AAVx/AAV9   <0.1 0.25-0.5  0.5-1  >1   Heart - EGFP+ AAVx/AAV9   <0.10.25-0.5  0.5-1  >1   Pancreas -EGFP+ AAVx/AAV9   <0.1 0.25-0.5 0.5-1  >1   *VG Biodistribution AAVx/AAV9 <1  1-10x   10-100x >100x  3). Toxicity Liver histopathology Histological grading  0 1 2 3 Brainhistopathology Histological grading  0 1 2 3 LFTs AAVx/Naïve  <1 1-2x 2-5x >5x  III. Selection of 2 CCVs from 3 LCVs and AAV9 in Chimpanzees(single dose, 3 animals/vector) Gene transfer Ch-A1AT-α-myc expAAVx/AAV9 >1 0.5-1   0.25-0.5 <0.1 Exp Stability  Day 7/Day 35   >0.750.5-0.75 0.25-0.5 <0.1 Immunology Capsid T Cells ELISPOT (sfu/10⁸cells)   <50  50-150  150-500 >500    Transgene T cells ELISPOT (sfu/10⁸cells)   <50  50-150  150-500 >500    Antibody to capsid AAVx/AAV9 <11-2x  2-5x >5x  Toxicity LFTs AAVx/AAV9 <1 1-2x  2-5x >5x  *VGBiodistribution: vector genome biodistribution is computed as the sum ofcopy number detected in spinal cord and Brain divided by the sum of copynumber detected in all other 9 tissues. The higher the ratio, more CNSrestricted.

In a first round of screening, two major aspects of vector biology(pre-existing immunity and gene transfer efficiency in the CNS) in 4parameters (in vitro NAB assay, in vivo adoptive transfer/livertransduction inhibition assay, EGFP gene transfer to the brain andspinal cord) are analyzed for all candidate vectors and compared withthose of AAV9 in both neonatal and adult mice. Three LCVs are selectedbased on the sum of all scores of 6 parameters with the best theoreticalscore equal to 0 and the worst theoretical score equal to 18 (in vitroNAB=3, passive transfer=3, Neonate brain EGFP=3, Neonate spinal cordEGFP=3, adult brain EGFP=3, and adult spinal cord EGFP=3) (Table 6).These LCVs are subjected to further analyses in mice and chimpanzees

In a second round of selection, LCVs at 3 different doses are evaluatedin two strains of neonatal and adult mice and compared to AAV9 in 13scoring categories. For the cell type tropisms in the mouse CNS, 3different cell type specific markers are used for identifying neurons,astrocytes and motor neurons (Table 7). Gene transfer efficiency isestimated by the percentage of EGFP positive cells in each cell type.The stability of gene transfer is scored by the ratio of percentages ofEGFP positive cells at days 14 and 90. The safety assessments in miceinclude brain and liver histopathology, LFTs, EGFP expression in liver,heart and pancreas, and biodistribution in 9 non-CNS tissues (lung,heart, spleen, liver, colon, kidney, pancreas, skeletal muscle andgonad). Since the vectors are delivered intravascularly to the CNS, thevector(s) with more limited biodistribution in non-CNS tissues aretypically the more favorable candidate(s). The lowest theoreticaccumulated score is zero and the highest is 39 for all 13 parameter.

TABLE 7 CNS Cell Type Specific Markers Cell Marker Antibody ManufacturerNeuron NeuN Mouse anti-NeuN Millipore Astrocyte GFAP Guinea Pig antiGFAP Advanced immunochemical Motor neuron ChAT Goat anti-ChAT Millipore(in spinal cord)

In additional to the phylogentic and physiological closeness to human,another reason to choose chimp as the systemic vector delivery model toselect CCVs from the LCVs is the chimp origin of those AAV9 variants.Evaluation of chimp AAV vectors in a chimp model is informative in manyaspects of the vector biology, particularly in the pre-existing B and Tcell immunity and their impact on the vector performance. The chimpstudy is focused on two aspects of vector biology: efficiency andstability of gene transfer and safety profiles. To mimic intravasculardelivery of the vectors to the CNS and study its impact on hostresponses, the vectors expressing chimpanzee α1-antitrypsin with a c-myctag in the C terminal (chA1AT-c-myc) is used and administeredintravenously at a dose of 1×10¹² GC/kg for noninvasive monitoring ofthis secreted reporter gene for gene transfer efficiency and stabilityin live animals without necropsy. Serum chA1AT-c-myc is measured atdifferent time points after gene transfer to the primary surrogatetissue liver (days 7, 14, 21, 28 and 35). In terms of safety profiles,at these same time points, blood samples are also taken for isolatingserum for liver function tests and peripheral blood mononuclear cells(PBMCs) for measuring capsid and chA1AT-c-myc specific T cells using theinterferon γ-Elispot assay. To study B cell response to vector capsid,neutralizing antibodies to corresponding AAV vectors in the serumsamples are analyzed by the in vitro NAB assay. LCVs are ranked based onthe sum of all scores of 6 parameters with the best theoretical scoreequal to 0 and the worst theoretical score equal to 18. CCVs areselected from LCVs and AAV9 based on their ranks in the performance intwo strains of mice and chimpanzees. These CCVs are further investigatedin neonatal and adult marmoset monkeys according to methods disclosedherein.

Screening of the Candidate Vectors for Pre-Existing B Cell Immunity inHumans

Pre-existing B cell immunity to vector capsid in gene therapy recipientsis a first immunological barrier to viral vector mediated gene transfer.Thus, B-cell immunity is a factor in clinical vector development and aselection criterion for vector biology evaluation. Commerciallyavailable clinical grade human IVIG is an Ig preparation pooled frommore than 60,000 random blood donors, which is considered to be a goodrepresentation of the existing B cell immunity to different AAVs thatare prevalent in human populations. This reagent is used in twodifferent assays to assess the potential of pre-existing immunity inhumans to inhibit gene transfer by those set of vectors. First, thevectors are screened using a well established transduction inhibitionassay which measures the reciprocal of the greatest dilution of humanimmunoglobulin (intravenous) (IGIV) that inhibits transduction (10).Secondly, the clones passed the in vitro neutralizing antibody (NAB)test are studied for the functional consequences of pre-existingimmunity in the passive transfer experiments in which pooled human Ig isinjected IV into CB6F1 hybrid mice 24 and 2 hours before IV injection ofEGFP expressing vector (1011 GCs per animal). Each group (N=5) receivesa different dose of human Ig—0, 0.12, 0.4, 1.2, 12 and 40 mg total dose.Assessment for the functionality of NAB to each vector is based on thelowest dose which demonstrates a significant inhibition of transductionas measured by EGFP transduction in liver at 28 days after genetransfer. Quantification of the expression levels of GFP in liver iscarried out as previously described (21). Inhibition at only high dosesof Ig indicates less interference of gene transfer and lower levels ofNAB. AAV9 vector serves as a control and bench marker for both the invitro and in vivo screening as described in Table 6.

Evaluation of the Candidate Clones in C57BL/6 Mice for the CNS GeneTransfer

A recent study by Foust et al presented evidence that cell type tropismsof AAV9 vector delivered to mouse CNS intravascularly are influenced bythe developmental stage of the CNS (9). Apparently, AAV9 targets neuronsefficiently in the CNS of neonates but astrocytes in the CNS of adults,which can be explained by the developmental changes in the nature of thecells surrounding the blood vessels in the CNS as well as the structureand molecular composition of the brain extracellular space (22).Considering potentially different applications in different CNSdisorders, these novel AAV9 variants are evaluated in both neonatal andadult C57BL/6 mice. AAV9 vector is included in the evaluation and itsperformance may serve as the bench marker for new vectors. For the firstround of vector screening, both neonatal (estimated body weight as 2grams) and adult (estimated body weight as 20 grams) mice receive thevectors at a dose of 1×10¹⁴ GCs/kg and are necropsied 2 weeks later toharvest the brain and spinal cord tissues for analysis. Specifically,one litter of single housed neonatal-day-1 pups is anesthetized andinjected with each vector through the temporal vein. The animals areeuthanized at 2 weeks after vector injection. The spinal cords andbrains are harvested and fixed. For the vector evaluation in adult mice,6 of 10 weeks old animals are infused with each vector via tail veininjections. The animals are euthanized 2 weeks later and transcardiallyperfused with 0.9% saline, then 4% paraformaldehyde. The spinal cordsand brains are harvested. For histological processing of the spinalcords and brains, the fixed tissues are transferred to a 30% sucrose andcryo-sectioned into 40μ thick sections for microscopic analysis. Inorder to perform semi-quantitative unbiased sterological assessment ofEGFP transduction in the CNS, the EGFP positive cells in 3 regions ofthe brain (retrosplenial/cingulate, dentate gyrus, and Purkinje cells)and the lumbar spinal cord is counted. The total numbers of EGFPpositive cells in each region are computed and compared to those in theAAV9 group. Three LCVs are selected from testing vectors based on theirserological reactivity to hIVIG as well as total numbers of EGFPpositive cells in either brain or spinal cord or all regions regardlesstheir cell types.

Further Characterization of LCVs and AAV9 in Two Strains of Mice

Differences in the gene transfer efficiency in the liver by AAV2 vectorbetween different mouse strains have been previously described (23).Vector biology of LCVs and AAV9 is evaluated in two different strains ofmice, C57BL/6 and BALB/c. Both of them are commonly used for AAV genetransfer studies. The evaluation is conducted in both neonatal and adultmice at several different vector doses: 0.3-, 1- and 3×10¹⁴ GC/kg. Theanimals are necropsied for analysis at days 14 and 90. The neonatalanimals used in this study are from litters of C57BL/6 and BALB/c Day-1neonates for each vector and each dose for necropsy at days 14 and 90.The adult animals are studied in the same way with 6 animals per vector,per dose and per time point. For this study, a total of 15 parametersare analyzed but they can be distilled down to 4 specific criteria:tropism and efficiency (% of EGFP positive neurons, motor neurons andastrocytes in the brain and spinal cord), stability of EGFP expressionin the brain and spinal cord (Day 90/Day14), non-CNS tissue targeting(EGFP expression in liver, heart and pancreas and the ratio of totalvector genome copies in the CNS and the sum of vector genomes detectedin all 9 non-CNS tissues) and vector related toxicity (brain and liverhistopathology and serum transaminase levels).

Evaluation of LCVs and AAV9 in Chimpanzees

Evaluation of AAV vectors in mice has contributed to vector discoveryand development, but vector performance in mice has not been alwayssuccessfully translated into that in large animal models, particularlyin vector related immune responses, perhaps due to the complexity of theadaptive immune response repertoire in higher primates (includinghumans) as compared with rodents. Chimpanzees are the closest relativeto humans genetically in the animal kingdom. Furthermore, chimps havebeen shown to share virus host ranges with humans to a greater extentthan any other species. This includes a variety of respiratory tractpathogens, such as respiratory syncytial virus (RSV), a number oflentiviruses, and hepatitis C virus. Chimpanzees are natural hosts oftranscriptionally active AAV9 variants. Therefore, chimps provide amodel system to study the immunological properties of primate AAVs,particularly those isolated from the chimpanzee. The data generated inchimps can predict safety and immunity in humans. LCVs and AAV9 vectorsthat express a secreted reporter gene are delivered intravenously to acohort of chimpanzees (n=3 per vector). Live animals are monitored forgene transfer, T cell responses to vector capsid proteins and totransgene product as well as antibody formation against vector capsid inblood samples. The animals are pre-screened for the presence of theneutralizing antibodies against the vector capsids. Those animals withundetectable NAB levels at the sensitivity of the NAB detection assayare enrolled for the study.

In order to dissect the capsid T cell response from that of thetransgene, the endogenous chimp α1-anti-tryspin gene is used to avoideliciting immune responses that could be caused specifically by crossingspecies. Another reason to use this secreted report is to noninvasivelymonitor the transgene expression in blood. This chimp gene has beenPCR-cloned. A c-myc tag is fused to the C terminal of the chimp-A1ATcDNA for detection by ELISA. The short c-myc-epitope is highly conservedacross species and should not be immunogenic, but is detectable withanti-c-myc antibody reaction in this ELISA. This strategy was usedsuccessfully with c-myc-tagged baboon AAT (24).

An Interferon-γ-ELISPOT assay may be used to detect both AAV capsid andtransgene specific T cells in chimp PBMCs. For these assays, peptidelibraries of the capsid proteins of LCVs and AAV9, and chimp-A1AT aresynthesized using standard techniques.

The decision on selecting CCVs is based on the data sets generatedin: 1) initial screening of clones by serological and gene transferassays, using AAV9 as the reference, to select LCVs; 2) furthercharacterization of LCVs and AAV9 in two strains of mice, and 3)evaluation of LCVs and AAV9 in the chimp model of systemic vectoradministration for the efficiency and stability of transgene expression,toxicity, activation of T cells to capsid and transgene product.Serological reactivity and total numbers of GFP positive cells iscompared in order to evaluate the pre-existing immunity to each vectorin a representative human population and the efficiency [total # of EGFP(+) cells in the CNS] of each vector in both neonatal and adult mice.The data are analyzed using the ANOVA method, where the serologicalreactivity and total numbers of EGFP positive cells are used as outcomevariables and vector types and age groups as the two predictive factors.With a subset of candidate vectors passing the serological tests invitro and in vivo, this experiment involves at least 2 factors, i.e.,the number of types of vector, and the number of age groups. Forexample, based on 7 vectors and 2 age groups, there are 7×2=14experimental groups, if each group has 6 experimental subjects (mice),data from 14×6=84 animals is used for this analysis. Potentialdifferential effects of vector type by age group are examined byincluding interaction (cross-product) term of vector type and age groupindicators in the ANOVA model. Relative contributions of vector type andage group to the total variations in the outcome measures are alsoevaluated. The results are used to identify optimal vector type and agegroup combinations.

Vector performance scores are also used to select LCVs; LCVs with thelowest scores and low variability in performance are typically selected.Selection is based on rating 15 parameters in 4-key criteria at a scaleof 0-3. ANOVA methods are used to analyze the data generated in thisphase. For example, this phase of the experiment may involve 5 factors,including vector type (3), doses of vector (3), strains (2), age groups(2), and time points (2). In this example, there are 3×3×2×2×2=72 uniquecombinations. If, for each combination, observations on 6 animals areavailable for analysis, in total, data on 72×6=452 animals are used forthis analysis. In order to test potential differential dose effectsbetween vector types, interaction terms (cross-product terms) betweenvector and dose variables are included in the ANOVA models. Significantinteraction terms imply possible existence of differential dose effectsby vector type. The results facilitate identification of optimal vectortype—dosage combinations. Relative contributions from the five factorsto the total variations in the performance scores are evaluated, whichprovide a basis for eliminating factors that are not important to thetotal performance in future experiments. The extent to which thevariances are homogenous across various levels of the five factors areevaluated. Any combinations with excessive variations are not consideredcandidate conditions in future experiments due to potentiallyunstable/unreliable experimental outcomes. The outcome variable isexpected to be normally distributed. The normality assumption for ANOVAanalysis appears realistic. In cases where the variable has a skeweddistribution (e.g., Poisson), a logarithmic transformation is applied tothe data.

Sample size and statistical power considerations: Inbreed mouse strainsare used for experiments, variation between animals under the samecontrolled experimental condition is considered to be very small, andthus 6 animals per experimental condition is justified. Power analysesare conducted as data is generated and proper adjustment to the groupsizes is made as necessary. Sample size for the chimpanzee study issmall due to restricted animal resources. ANOVA methods are not alwaysapplicable. In some cases, we provide only descriptive statisticswithout formal statistical testing.

Further Characterization of Intravascularly Delivered CCVs for the CNSGene Transfer in Marmoset Monkeys

Structural differences among novel AAV9 variants leads to theirfunctional differences in neuronal and neuroglial cell tropism,efficiency and stability of CNS transduction, vector related toxicity,and immunological properties in NHPs. Systemic delivery of LCVs tochimpanzees provides critical data to assess the vector toxicity, the Tcell immunities towards both vector capsids and transgene as well as theB cell immunities against different vector capsids. Intravasculardelivery of a secreted reporter gene transfer in chimpanzees alsoenables an evaluation of gene transfer efficiency and stability in thesurrogate tissue (primarily liver). Vector candidate(s) suitable forclinical development of the CNS directed gene therapy, are identified byevaluating the candidate vectors in a NHP CNS gene transfer model.Marmoset monkeys are useful as the model for the CNS gene transfer.Found originally in the forests of South America, marmoset monkeys arethought to be phyletic dwarfs that have evolved from a larger ancestor.They have many genetic and physiological similarities to humans. Thecommon marmoset (Callithrix jacchus) has several advantages as anexperimental animal model. The small size of the common marmoset(approximately 300-600 g) reduces the vector production burdenremarkably and makes it easier to handle relative to larger Old Worldmonkeys. As laboratory animals, marmosets also have reduced cost andsafety hazards in comparison to Old World monkeys. They can live up to15 years in captivity, with breeding pairs giving birth to twins (about100 grams each) every 5 months (25). Therefore, these marmosets can beeasily reproduced in the laboratory setting, making possible supply oflarge numbers of marmosets with consistent microbiological and geneticquality. Marmoset monkeys have been used to investigate a variety ofCNS-related diseases, especially for the research of Parkinson's diseaseand Huntington's disease, as summarized in a review paper (26). In fact,marmosets are the one of the most extensively used NHP models inbiomedical research. The marmoset study is carried out in both neonataland adult animals. CCVs are evaluated by 18 measurements in 5categories: tropism and efficiency in the CNS, stability in the CNS,non-CNS tissue targeting, immune responses and toxicity, using a scoringsystem similar to that used for selection of the CCVs in mice andchimps.

Experimental Design

CCVs are extensively evaluated in marmosets for cell type tropism, genetransfer efficiency and stability in the CNS, non-CNS tissue targetingand gene expression, CNS and liver pathology, transgene and capsidimmunities and vector related toxicity as summarized in the Table 8.

TABLE 8 Study Design for Marmoset Monkey Experiments Rating Age/animal#Parameter Measurement 0 1 2 3 Postnatal-Day −1 (20 total) 1. Tropism andefficiency n = 4 Spinal cord % EGFP(+)/ChAT(+)    >75 75-50  50-25 <25per vector (3 × 10¹⁴ GC/kg) % EGFP(+)/GAFP(+)   >75 75-50  50-25 <25 pertime point (Days 7 and 35) Brain % EGFP(+)/NeuN(+)    >75 75-50  50-25<25 n = 4, PBS control % EGFP(+)/GAFP(+)   >75 75-50  50-25 <25 2.EGFP - stability in CNS Adult (20 total) Spinal cord: Motor N Day 7/Day35 >0.75 0.5-0.75 0.25-0.5    <0.25 n = 4 Astrocytes Day 7/Day 35 >0.750.5-0.75 0.25-0.5    <0.25 per vector (3 × 10¹⁴ GC/kg) Brain: Neuron Day7/Day 35 >0.75 0.5-0.75 0.25-0.5    <0.25 per time point (Days 7 and 35)Astrocytes Day 7/Day 35 >0.75 0.5-0.75 0.25-0.5    <0.25 n = 4, PBScontrol 3. Non-CNS tissue targeting Transduction Liver % EGFP(+) <1010-30  30-60 >60 Heart % EGFP(+) <10 10-30  30-60 >60 Pancreas % EGFP(+)<10 10-30  30-60 >60 VG Biodistribution  GC, CNS/GC, 9 tissues >10⁻²>10⁻³ >10⁻⁴ >10⁻⁵ 4. Immune responses Cap T cells ELISPOT (sfu/10⁶ cells<50 50-150 150-500 >500  EGFP T cells ELISPOT (sfu/10⁶ cells <50 50-150150-500 >500  NAB to vector cap Reciprocal titer <1/10²  <1/10³  <10⁴ >10⁵  5. Toxicity brain pathology Histological grading 0 1 2  3 liverhistopathology Histological grading 0 1 2  3 LFTs  PBS/vector <1 1-2x 2-5x  >5x

Assessment of CCVs in Neonatal and Adult Marmoset Monkeys for CNS GeneTransfer

CCVs are further investigated in both neonatal and adult marmosets. Theadult animals are pre-screened for neutralizing antibodies against CCVsby the in vitro transduction inhibition assay. The neonatal animals areproduced by programmed pregnancy. The mothers are identified forprogrammed pregnancy based on their neutralizing antibody titers to thevectors. The pregnant mothers are tested again for the NAB titers 2weeks before the due dates. The highest vector dose (3×10¹⁴ GC/kg) usedfor the neonatal and adult mice is the vector dose for both neonatal andadult marmosets. For the neonatal study, a total of 20 animals areenrolled to evaluate CCVs. Four out of 20 animals receive PBS injectionand serve as the controls. Eight postnatal-day-1 animals are infusedwith each of CCVs, out of which 4 animals each is necropsied at days 7and 35 for analysis. A total of 20 adult male animals are used in thisstudy in a design identical to that of the neonatal marmosets. Spinalcord and brain tissues are harvested at the necropsies. The fixedtissues are cryo-sectioned and subjected to immunofluorescent stainingfor the cell markers listed in the Table 3 to label neurons, astrocytesand motor neurons followed by microscopic examination. Percentages ofEGFP positive cells in each cell types indifferent regions of the brainand spinal cord at different time points are computed to assess celltypetropism, spreading and stability of the EGFP gene transfer by 2CCVs.

Capsid and Transgene Immunities

Analyses of capsid and transgene T cells are performed on both PBMCsduring the live phase of the marmoset studies and lymphocytes isolatedfrom different lymphoid tissues at the necropsies. Below is a list ofPBMCs collected at different time points and tissues collected at thenecropsies.

-   -   PBMCs: Day 7 prior to vector injection, days, 14, 28, and 35        post vector infusion    -   Tissue lymphocytes: Liver, peritoneal lavage, spleen, and        axillary, inguinal, iliac and mesenteric lymph nodes

For the neonatal animals, pre-vector PBMCs are sampled at the time ofinjection. IFN-γ ELISPOT assays are carried out for all of those samplesto detect capsid and EGFP specific T cells. At each bleed, serum samplesare also collected for the NAB assay to determine the B cell responsesto the viral vector capsids.

Vector Related Toxicity and Non-CNS Bio-Distribution

During the live phase of the study and the time of necropsy, serumtransaminase levels (LFTs for ALT and AST) of the study animals aremonitored to investigate potential vector related liver toxicity. At thetime of necropsy, a part of brain and liver tissues are fixed, paraffinembedded, sectioned and stained for histopathology study. To inspectEGFP transduction in non-CNS tissues, liver, heart and pancreas tissuesare collected for fixation, cryo-section and microscopic examination.Finally, in addition to the brain and spinal cord, a panel of 9 tissues(lung, heart, spleen, liver, colon, kidney, pancreas, skeletal muscleand gonad) is collected at the necropsy for real time PCR quantificationof the persisted vector genomes.

Statistical Analysis and Alternative Strategies

Vector type (e.g., 2), age groups (e.g., 2), and time points (e.g., 2)are 3 main factors involved in this experiment. Those factors aretranslated into, e.g., 2×2×2=8 unique combinations, each of which has 4experimental animals. In total, data on, e.g., 8×4=32 animals isavailable for the statistical analysis. In this study, 4 animals thatreceive PBS from each age group serve as the baseline to better assessthe vector related toxicity. ANOVA methods are used for data analysis.The outcomes of the study are informative for deciding which CCVs shouldbe advanced for clinical application. A concern over the CNS targetingof gene therapeutics through systemic vector delivery is the potentiallyharmful consequences of high levels of ectopic expression of the CNSgenes in other tissues. The extent to which identified CCVs have CNSrestricted biodistribution is addressed by employing the CNS specificpromoter or microRNA regulation or both in our expression cassettes tode-target transgene expression from other tissues and cell types,particularly those involved with cellular immunity. Both strategies havebeen used to de-target the liver from transgene expression (27).

REFERENCES FOR EXAMPLE 5

-   1. Fu H, Muenzer J, Samulski R J, et al. Self-complementary    adeno-associated virus serotype 2 vector: global distribution and    broad dispersion of AAV-mediated transgene expression in mouse    brain. Mol Ther 2003; 8:911-7.-   2. Gao G, Lu Y, Calcedo R, et al. Biology of AAV serotype vectors in    liver-directed gene transfer to nonhumanprimates. Mol Ther 2006;    13:77-87.-   3. Manno C S, Pierce G F, Arruda V R, et al. Successful transduction    of liver in hemophilia by AAV-Factor IX andlimitations imposed by    the host immune response. Nat Med 2006; 12:342-7.-   4. Mandel R J, Manfredsson F P, Foust K D, et al. Recombinant    adeno-associated viral vectors as therapeutic agents to treat    neurological disorders. Mol Ther 2006; 13:463-83.-   5. Gao G P, Alvira M R, Wang L, Calcedo R, Johnston J, Wilson J M.    Novel adeno-associated viruses from rhesus monkeys as vectors for    human gene therapy. Proc Natl Acad Sci USA 2002; 99:11854-9.-   6. Gao G, Alvira M R, Somanathan S, et al. Adeno-associated viruses    undergo substantial evolution in primates during natural infections.    Proc Natl Acad Sci USA 2003; 100:6081-6.-   7. Gao G, Vandenberghe L H, Alvira M R, et al. Clades of    Adeno-associated viruses are widely disseminated in human tissues. J    Virol 2004; 78:6381-8.-   8. Gao G, Vandenberghe L H, Wilson J M. New recombinant serotypes of    AAV vectors. Curr Gene Ther 2005; 5:285-97.-   9. Foust K D, Nurre E, Montgomery C L, Hernandez A, Chan C M, Kaspar    B K. Intravascular AAV9 preferentially targets neonatal neurons and    adult astrocytes. Nat Biotechnol 2009; 27:59-65.-   10. Calcedo R, Vandenberghe L H, Gao G, Lin J, Wilson J M. Worldwide    epidemiology of neutralizing antibodies to adeno-associated viruses.    J Infect Dis 2009; 199:381-90.-   11. Arbetman A E, Lochrie M, Zhou S, et al. Novel caprine    adeno-associated virus (AAV) capsid (AAV-Go.1) is closely related to    the primate AAV-5 and has unique tropism and neutralization    properties. J Virol 2005; 79:15238-45.-   12. Schnepp B C, Jensen R L, Chen C L, Johnson P R, Clark K R.    Characterization of adeno-associated virus genomes isolated from    human tissues. J Virol 2005; 79:14793-803.-   13. Carter B J, P. Trempe J, Mendelson E. Adeno-associated virus    gene expression and regulation. In: Tijssen P, editor. Handbook of    parvoviruses. Boca Raton: CRC Press Inc.; 1990. p. 227-54.-   14. Vandenberghe L H, Wilson J M, Gao G. Tailoring the AAV vector    capsid for gene therapy. Gene Ther 2009; 16:311-9.-   15. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular    Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol    Evol 2007; 24:1596-9.-   16. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL    workspace: a web-based environment for protein structure homology    modelling. Bioinformatics 2006; 22:195-201.-   17. Mingozzi F, Maus M V, Hui D J, et al. CD8(+) T-cell responses to    adeno-associated virus capsid in humans. Nat Med 2007; 13:419-22.-   18. Akache B, Grimm D, Pandey K, Yant S R, Xu H, Kay M A. The    37/67-kilodalton laminin receptor is a receptor for adeno-associated    virus serotypes 8, 2, 3, and 9. J Virol 2006; 80:9831-6.-   19. Fisher K J, Gao G P, Weitzman M D, DeMatteo R, Burda J F, Wilson    J M. Transduction with recombinant adenoassociated virus for gene    therapy is limited by leading-strand synthesis. J Virol 1996;    70:520-32.-   20. Roy S, Gao G, Lu Y, et al. Characterization of a family of    chimpanzee adenoviruses and development of molecular clones for gene    transfer vectors. Hum Gene Ther 2004; 15:519-30.-   21. Scallan C D, Jiang H, Liu T, et al. Human immunoglobulin    inhibits liver transduction by AAV vectors at low AAV2 neutralizing    titers in SCID mice. Blood 2006; 107:1810-7.-   22. Lowenstein P R. Crossing the rubicon. Nat Biotechnol 2009;    27:42-4.-   23. Wang L, Calcedo R, Nichols T C, et al. Sustained correction of    disease in naive and AAV2-pretreated hemophilia B dogs:    AAV2/8-mediated, liver-directed gene therapy. Blood 2005;    105:3079-86.-   24. Song S, Scott-Jorgensen M, Wang J, et al. Intramuscular    administration of recombinant adeno-associated virus 2 alpha-1    antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model:    safety and immunologic aspects. Mol Ther 2002; 6:329-35.-   25. Abbott D H, Barnett D K, Colman R J, Yamamoto M E,    Schultz-Darken N J. Aspects of common marmoset basic biology and    life history important for biomedical research. Comp Med 2003;    53:339-50.-   26. Mansfield K. Marmoset models commonly used in biomedical    research. Comp Med 2003; 53:383-92.-   27. Xie J, Su Q, Xie Q, Mueller C, Zamore P, Gao G. MicroRNA    regulated tissue specific transduction by rAAV vector. American    Society of Gene Therapy Annual Meeting. San Diego; 2009.

Example 6: Sensitive Detection and Characterization of AAV Contaminationin Producer Cell Lines for Recombinant Biologics

AAV viruses are very stable and easily aerosolized. The life cycle ofAAV consists of two stages: a latent stage and a replication stage. Inthe absence of adenovirus, AAV infects host cells in a very stealthymanner to establish its latency. Early studies with wild type AAVinfection of cell lines in vitro suggested that AAV site-specificallyintegrated into human Chromosome 19 q 13, although, as yet, no directevidence supports the site specific integration of AAV in vivo. Onlywhen helper functions are provided by co- or subsequent infection ofAdenovirus or HSV are the latent AAV genomes rescued, replicated andpackaged into infectious virions. The primary adenovirus genes withhelper functions for AAV replication and packaging include E1a, E1b,E2a, E4 and VARNA. However, extensive studies with recombinant AAV, inthe absence of selective pressure, have revealed that rAAV genomes arecolonized in the host nuclei primarily as the episomal forms such ashigh molecular weight linear concatomers, and circular monomer andconcatamers.

Due to its capability of being aerosolized and existing as a latent andpersistent infection, contamination of commonly used cell lines by AAVis becoming a major issue for the cell line based GMP manufacturing ofrecombinant biologics. Some major challenges in the detection andcharacterization of stealthy infection of cell lines by AAV include thelack of any microscopically visible pathological effect, primarilyepisomal persistence of AAV genomes that leads to a dilutional effectafter cell divisions and inconsistency in their detection, and a highrate of AAV contamination in adenovirus preparations when used as helpervirus for AAV rescue studies. When PCR based molecular methods areemployed for the rescue of AAV sequences from testing cell lines,several technical obstacles negatively impact the detection andcharacterization of endogenous AAV proviral sequences. First, in theseveral contaminated cell lines which were tested, AAV sequences existin very low abundance (ranging from 0.1 to 0.001 copy per diploidgenome), which requires highly efficient and reliable PCR methodologyand technical skills to accomplish the sequence amplification. Second,the primer design represents another challenge. Rep genes are usuallyhighly conserved and good targets for primer design and PCR detection.But high sequence homology between rep genes of different AAV serotypesmakes serotype identification of different AAVs difficult. On the otherhand, the natural diversity of the AAV family is primarily displayed assignificant sequence variations in AAV cap genes, which makes diagnosisof AAV identity relatively easy. This kind of target sequence variationsalso results in difficulties in primer design for PCR amplification ofcap genes.

Producer cell clones are examined to detect contamination with low copynumbers of known, or unknown, species of AAV, which persist as episomalproviral genomes and may be transcriptionally active in a latent state.Isolation and characterization of the AAVs may be accomplished throughany one or a combination of the following approaches. For example, AAVproviral genomic sequences may be assessed by signature PCRs, cloning(e.g., TOPO cloning), sequencing and bioinformatic analysis. AAV viralRNA transcripts in the contaminated cell clones may be assessed byRT-PCR, cloning, sequencing and bioinformatic analysis. AAV proviralsequences in the contaminated cells may be rescued by providingadenovirus helper functions to the contaminated producer cell clone(s).

Analysis of AAV Proviral Genomic Sequences Using Signature PCR:

Detection of AAV proviral genomic sequences using short signature PCRachieves a detection limit of 1-10 copies of AAV cap sequence in thebackground of mouse liver total cellular DNA. Short signature PCR uses aprimer set that can anneal to and amplify a signature region (about 260bp) in the hypervariable region 2 of VP1 of most of AAV cap gene.

Detection of AAV proviral genomic sequences using long signature PCRachieves a detection limit of 10 copies of AAV cap sequence in thebackground of mouse liver total cellular DNA. Long signature PCR usingprimer sets are used that can anneal to and amplify a genomic region(spanning 800 bp of rep and 2.2 kb of entire cap VP1 gene or just 2.2 kbof the entire cap VP1 gene) of most of AAV serotypes and genotypes.

Protocol:

-   -   Detection, isolation and characterization of AAV proviral        sequences in the testing articles by signature PCR, Topo        cloning, sequencing and bioinformatic analysis.    -   1). Testing articles and controls:        -   a. Testing articles: cellular DNAs from AAV strong positive,            weak positive and negative cell clones        -   b. Positive control: Detroit 6-7374 cells. This is AAV            latently infected Detroit 6 cell line which has 0.1-1 copy            per cell of latent AAV genome (Bern K I et al, 1975,            Virology 68(2):556-560; Kotin R M and Berns K I, 1989,            Virology 170(2): 460-467; Kotin R M et al., 1990, J. Virol.            87-2211-2215 and Gao et al., unpublished data).        -   c. Negative controls: total cellular DNAs from original ATCC            293 cells, Invitrogen 293 cells and naïve mouse liver.        -   d. pAAV2rep/cap plasmid is used to spike into total cellular            DNA of either AAV free 293 cells or naïve mouse liver as            copy number references to monitor the sensitivity of the PCR            detection.    -   2). Primer sets used:        -   AV2as/AV1 ns (3 kb rep+cap)        -   AV2as/19S (1.6 kb cap)        -   CapF/AV2cas (2.2 kb cap)        -   19S/18as (0.26 kb cap)        -   Rep3/rep5 (0.44 kb rep)    -   3). PCR and cloning reagents:        -   Cellular DNA extraction kit        -   DNase/RNase free water        -   PCR kit        -   PCR purification kit        -   Gel purification kit        -   Topo cloning kit        -   Competent cells        -   Ligation and other cloning reagents        -   Various restriction enzymes        -   Antibiotics for cloning        -   DNA molecular weight markers    -   4). Outline of the experiment:

(1). Assay development. To optimize PCR conditions with different primersets using various copy numbers of pAAV2rep/cap plasmid DNA as well astheir counterparts spiked into cellular DNA of either clean 293 cells ormouse liver to determine sensitivity of the PCR assay and potentialinterference of cellular DNA to AAV target specific detection.

(2). Workflow. Various cell clones of testing article as well aspositive and negative controls; total cellular DNA extraction; performPCR together with copy number reference controls spiked into AAV freecellular DNA; gel electrophoresis; cloning PCR positive fragments;restriction mapping analysis for gross identification; sequence analysisand bioinformatic analysis to confirm the identity.

Analysis of AAV Proviral RNA Transcript Sequences Using RT-PCR:

AAV viral RNA transcripts are analyzed by combining reversetranscription reaction with the highly sensitive PCR designs (short andlong signature PCR) described above.

Protocol:

Detection, isolation and characterization of AAV viral RNA transcriptsin the contaminated cell clones.

1). Testing articles and controls:

a. Testing articles: AAV strong positive, weak positive and negativecell clones based on the data from contracted testing labs.

b. Positive control: Detroit 6-7374 cells. This is AAV latently infectedDetroit 6 cell line which has 0.1-1 copy per cell of latent AAV genome(Bern K I et al, 1975, Virology 68(2):556-560; Kotin R M and Berns K I,1989, Virology 170(2): 460-467; Kotin R M et al., 1990, J. Virol.87-2211-2215 and Gao et al., unpublished data).

c. Negative controls: Original ATCC 293 cells, Invitrogen 293 cells andnaïve mouse liver.

d. pAAV2rep/cap plasmid is used to spike into total cellular DNA ofeither AAV free 293 cells or naïve mouse liver as copy number controlsto monitor the sensitivity of the PCR detection.

e. Infectious clones of pAdS plasmid is used for activating latent AAVtranscription in the positive control Detroit 6-7374 cells, while pAdhelper plasmid is transfected into the 293 cell-based testing cellclones for initiating viral RNA transcription of contaminating AAVsequences.

2). Primer sets used for conventional PCR and RT-PCR:

-   -   AV2as/19S (1.6 kb cap)    -   CapF/18as (700 bp cap)    -   CapF/AV2cas (2.2 kb cap)    -   19S/18as (0.26 kb cap)    -   Rep3/Rep5 (0.44 kp rep)

3). Primer and probe sets used for real time PCR and RT-PCR:

-   -   Rep1F/Rep1R    -   Cap1F/Cap1R

4). Tissue culture, RNA extraction, RT-PCR and cloning reagents:

-   -   Standard tissue culture reagents    -   DNA transfection reagents    -   Trizol    -   Qiagen RNAeasy kits    -   RNase free-DNase I    -   RNase Zap    -   DNase and RNase free water    -   High capacity reverse transcription kit    -   One step qRT-PCR kit    -   Real time PCR master mix    -   Real time PCR primer/probe sets    -   Conventional PCR reagent    -   Topo cloning kit    -   Ligation and other cloning reagents    -   Various restriction enzymes    -   Antibiotics for cloning    -   DNA and RNA molecular weight Markers

4). Outline of the experiment

The positive control Detriot-6-7374 cells, AAV free-negative controlcells and testing cell clones are cultured with or without transfectionof adenovirus helper plasmids for 48 hours. Total cellular RNA isprepared from each culture with or without DNase treatment. Theconventional and real time RT-PCR reactions are set up for each samplethat has undergone the following treatments.

a. RNA samples not treated with DNase I

b. RNA samples treated with DNase I but not with reverse transcriptase

c. RNA samples treated with both DNase I and reverse transcriptase

The PCR products are cloned, characterized by restriction mapping andpositive clones are sequenced and subjected to bioinformatic analysisfor the identity. Furthermore, relative transcriptional activity of AAVviral genes in the testing cell clones in the presence and absence ofadenovirus helper functions are quantified by one step qRT-PCR.

Viral Rescue Assay of AAV Proviral Genomes

For viral rescue of AAV proviral genomes in contaminated producer cellclone(s), adenovirus helper functions are provided by transfecting an Adhelper plasmid that contains all essential helper genes but could notproduce any infectious adenovirus. This strategy has the followingunique advantages. First, providing helper function by plasmid, not thevirus, essentially eliminates the possibility of AAV contaminants thatcould be brought into the testing process by adenovirus infection.Second, the absence of adenovirus replication and cytopathic effectspromotes rescue and replication of AAV in the contaminated cellclone(s).

The sensitivity and reliability of the assay is enhanced by serialpassage of crude lysates of the testing article onto either the testingarticle(s) themselves or clean 293 cells to accomplish biologicalamplifications of the target signal.

The biological amplified target sequences are isolated, characterizedand quantified by the highly sensitive and reliable conventional andreal time PCR and RT-PCR assays described above. The combination ofbiological amplification and efficient PCR amplification furtherimproves the sensitivity of the assay and make it possible to detectvery low abundance of AAV sequences in the contaminated cell clones.Particularly, the primer and probe sets for real time PCR quantificationof AAV sequences that exist in the original testing article as well asthat are biologically amplified in different passages are designed totarget the conserved sequences in the AAV genome.

Protocol:

Rescue and characterization of AAV proviral genomes in the contaminatedproducer cell clone(s).

1). Testing articles and controls:

a. Testing articles: AAV strong positive, weak positive and negativecell clones based on the data from contracted testing labs.

b. Positive control: Detroit 6-7374 cells. This is AAV latently infectedDetroit 6 cell line which has 0.1-1 copy per cell of latent AAV genome(Bern K I et al, 1975, Virology 68(2):556-560; Kotin R M and Berns K I,1989, Virology 170(2): 460-467; Kotin R M et al., 1990, J. Virol.87-2211-2215 and Gao et al., unpublished data).

c. Negative controls: Original ATCC 293 cells, Invitrogen 293 cells andnaïve mouse liver.

d. pAAV2rep/cap plasmid is used to spike into total cellular DNA ofeither AAV free 293 cells or naïve mouse liver as copy number controlsto monitor the sensitivity of the assay.

e. Infectious clones of pAdS plasmid are used for activating latent AAVtranscription in the positive control Detroit 6-7374 cells, while pAdhelper plasmid is transfected into the 293 cell-based testing cellclones for initiating viral RNA transcription of contaminating AAVsequences.

f. Wild type AAV2 with known infectious titer for assay development.

2). Primer sets used for conventional PCR and RT-PCR:

-   -   AV2as/AV1 ns (3 kb rep+cap)    -   AV2as/19S (1.6 kb cap)    -   CapF/18as (700 bp cap)    -   CapF/AV2cas (2.2 kb cap)    -   19S/18as (0.26 kb cap)    -   Rep3/Rep5 (0.44 kb rep)

3). Primer and probe sets used for real time PCR and RT-PCR:

-   -   Rep1F/Rep1R    -   Cap1F/Cap1R

4). Tissue culture, RNA extraction, RT-PCR and cloning reagents:

-   -   Standard tissue culture reagents    -   DNA transfection reagents    -   Trizol    -   Qiagen RNAeasy kits    -   RNase free-DNase I    -   RNase Zap    -   DNase and RNase free water    -   High capacity reverse transcription kit    -   One step qRT-PCR kit    -   Real time PCR master mix    -   Real time PCR primer/probe sets    -   Conventional PCR reagents    -   Topo cloning kit    -   Ligation and other cloning reagents    -   Various restriction enzymes    -   Antibiotics for cloning

5). Outline of the experiment

-   -   (1). Production of wild type AAV2 virus as the positive control        for assay development. pSub201, an infectious molecular clone of        AAV2, is co-transfected with pAd helper plasmid into 293 cells        for AAV2 rescue and packaging. The virus is purified by 3 rounds        of gradient centrifugation. The infectious titer of this wild        type positive control AAV is determined by serial        dilution/infection and real time PCR based TC-ID50 assay.    -   (2). Assay development and validation to determine the        sensitivity and reproducibility of the infection/passage assay.        AAV free-293 cells seeded in 60 mm or 100 mm plates are infected        with AAV2 control virus at MOIs of 0, 1, 10, 100 and 1000 in 6        replicates. For 3/6 replicates of each infection, cells are        transfected with an infectious molecular clone of Ad5 (pAdS) at        3 hours after the infection. All replicates are harvested at 7        days post-infection and equally divided into 2 parts. One part        of each is used for DNA and RNA extraction and real time PCR and        RT-PCR analysis. The other part of each cell pellet is        re-suspended in 10 mM Tris, pH8.0 and subjected to 3 cycles of        freeze/thaw. One third of each lysate is used to passage onto        the 2nd sets of 293 cells without additional transfection of        pAdS plasmid. Four days after infection, the cells are harvested        and processed as above towards a 3rd passage. Unused viral        lysate is stored at −80° C. DNA and RNA samples from each        passages are assayed by real time PCR and RT-PCR for        quantification. Conventional PCR and RT-PCR is carried out, as        needed, for AAV sequence isolation, cloning and sequence        confirmation.    -   (3). Rescue of latent AAV2 from positive control Detroit 6-7374        cells. The cells are seeded in 60 mm or 100 mm plates in 6        replicates. Three of 6 plates are transfected with infectious        molecular clone of ad5, pAdS. One week after transfection, the        cells is processed and analyzed as described in (2). This will        serve as the positive control experiment to demonstrate the        capability to rescue and amplify the AAV2 genome in a latent        infected cell line by this rescue/passage assay.    -   (4). Examination of the testing producer cell clones for        rescueable AAV provirus contamination. The experiment is carried        out as described in (1) and (2) except for the following        differences.    -   a. the conventional PCR and RT-PCR is performed for AAV sequence        isolation, cloning, sequencing and bioinformatic analysis to        identify the contaminating AAV in the testing cell clones.    -   b. As an option, if there is indeed rescueable and amplifiable        AAV contamination in the producer cell clones, the entire        proviral genome may be cloned (from ITR to ITR) as an infectious        molecular clone for further characterization.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thisdescription or illustrated in the drawings. The invention is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is:
 1. A recombinant adeno-associated virus (rAAV) comprising a nucleic acid encoding a heterologous transgene and an AAV capsid, wherein the AAV capsid comprises a protein comprising an amino acid sequence selected from SEQ ID NOs.: 96, 98, 100, and
 111. 2. The rAAV of claim 1, wherein the heterologous transgene comprises a tissue-specific promoter operably linked to a coding sequence.
 3. The rAAV of claim 2, wherein the coding sequence encodes a therapeutic protein.
 4. The rAAV of claim 3, wherein the therapeutic protein is aromatic amino acid decarboxylase (AADC).
 5. A composition comprising the rAAV of claim
 1. 6. The composition of claim 5, further comprising a pharmaceutically acceptable carrier.
 7. The rAAV of claim 1, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 96. 8. A composition comprising the rAAV of claim 7 and a pharmaceutically acceptable carrier.
 9. The rAAV of claim 1, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 98. 10. A composition comprising the rAAV of claim 9 and a pharmaceutically acceptable carrier.
 11. The rAAV of claim 1, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 100. 12. A composition comprising the rAAV of claim 11 and a pharmaceutically acceptable carrier.
 13. The rAAV of claim 1, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 111. 14. A composition comprising the rAAV of claim 13 and a pharmaceutically acceptable carrier.
 15. The rAAV of claim 3, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 96. 16. The rAAV of claim 3, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 98. 17. The rAAV of claim 3, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 100. 18. The rAAV of claim 3, wherein the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO.:
 111. 